Monoclonal antibodies against loricrin

ABSTRACT

The present invention relates inter alia to the development of monoclonal antibodies against Loricrin and methods for determining whether a subject with melanoma has an increased risk of metastasis using said antibodies.

FIELD OF THE INVENTION

The present invention relates inter alia to the development ofmonoclonal antibodies against Loricrin and methods for determiningwhether a subject with melanoma has an increased risk of metastasisusing said antibodies.

BACKGROUND TO THE INVENTION

Following diagnosis with melanoma, patient prognosis is presently basedon clinico-pathological criteria within the primary tumour. Althoughrobust, AJCC staging criteria can only broadly categorise risk ofdisease progression based on epidemiological data from cohorts of otherpatients with similar staging in prior clinical reviews.

Currently, patients with thin melanomas are categorised as AJCC stageIa/b and considered as being at a low risk of metastasis. However, 10%of MCC Ia/b patients develop distant disease, and as such aremis-diagnosed based on current disease stratification. The ability tostratify this group of seemingly ‘low-risk’ patients more accuratelywould have a major impact on patient follow-up, the intensity ofinvestigation, and the instigation of targeted adjuvant therapies in atrial or therapeutic setting.

Loricrin is a glycine-serine-cysteine-rich protein encoded by the LORgene. In humans, the LOR gene is part of a cluster of genes onchromosome 1 called the epidermal differentiation complex. These genesare involved in the formation and maintenance of the outer layer of skin(the epidermis), particularly its tough outer surface (the stratumcorneum). The stratum corneum, which is formed in a process known ascornification, provides a sturdy barrier between the body and itsenvironment. Each cell of the stratum corneum, called a corneocyte, issurrounded by a protein shell called a cornified envelope (CE). Loricrinis the predominant protein of the cornified envelope. Links betweenLoricrin and other components of the envelopes hold the corneocytestogether and help give the stratum corneum its strength.

Immunohistochemical expression of epidermal Loricrin has been identifiedas a biomarker for disease progression in melanoma (WO 2016/075440 A1).However, there remains a need to improve prognosis of metastasis insubjects suffering from melanoma. There also remains a need to improvetreatment of patients suffering from melanoma and to decrease thelikelihood of progression to metastasis.

It is an aim of some embodiments of the present invention to at leastpartially mitigate some of the problems identified in the prior art.

SUMMARY OF CERTAIN EMBODIMENTS OF THE INVENTION

Certain aspects of the present invention relate to the development ofmonoclonal antibodies against Loricrin and methods of using the same.

Antibodies described herein allow the improved detection of Loricrin ascompared to known research grade polyclonal antibodies (AbcamBiochemicals, Cambridge, UK; 24722). In particular, the anti-Loricrinantibodies described herein have improved specificity to Loricrin ascompared to research grade antibodies. Unexpectedly, these anti-Loricrinantibodies provide superior specific staining of uppermost terminallydifferentiated keratinocytes within the epidermis. In contrast, researchgrade polyclonal antibodies against loricrin stain several layers ofdifferentiated keratinocytes.

The loss of Loricrin staining in the most differentiated top layer ofkeratinocytes in the stratum corneum of the epidermis is associated withan increased risk of tumor spread. The accurate assessment of Loricrinexpression is important in determining metastatic risk. The superiorspecific staining of the antibodies described herein may allow moreaccurate assessment of loss of Loricrin expression as compared toresearch grade antibodies. For example, inaccurate detection usingresearch grade antibodies against Loricrin may lead to the risk of asubject erroneously being determined as having an increased risk ofmetastasis. In such a scenario, the subject may undergo unnecessarypsychological distress and/or medical procedures when in fact thesubject is at a low risk of metastasis. Use of the antibodies describedherein aims to mitigate such risks. Use of the antibodies describedherein provides improved ease, accuracy and speed of determiningLoricrin expression.

Fully human, recombinant monoclonal antibodies were generated using afull-length recombinant sequence of Loricrin and Human CombinatorialAntibody Libraries (HUCAL, Bio-Rad). Importantly, the antibodiesdescribed herein can be produced identically by sequence, securingconsistent and reliable antibody production for clinical in vitrodiagnostic use.

Further antibodies are also described herein that allow improveddetection of Ambra-1 as compared to known research grade polyclonalantibodies (Abcam Biochemicals, Cambridge, UK; 69501). Unexpectedly,these anti-Ambra-1 antibodies have improved specificity to Ambra-1 ascompared to research grade antibodies. Unexpectedly, these anti-Ambra-1antibodies provide superior staining of epidermal keratinocytes in linewith their differentiation status. The anti-Ambra-1 antibodies describedherein also show superior specific staining of melanoma tumor cells ascompared to the research grade antibodies. Thus, the anti-Ambra-1antibodies described herein provide improved ease, accuracy and speed ofdetermining Ambra-1 expression.

The loss of Ambra-1 staining in the epidermal keratinocytes isassociated with an increased risk of tumor spread. The loss of Ambra-1is due to a loss of terminal differentiation of keratinocytes. Theaccurate assessment of Ambra-1 expression is important in determiningmetastatic risk. The superior specific staining of the antibodiesdescribed herein allows more accurate assessment of loss of Ambra-1expression as compared to research grade antibodies.

The more consistent and distinct pattern of expression of theanti-Loricrin (and optionally anti-Ambra-1) antibodies described hereinfacilitates rapid and accurate identification of those subjects havingdecreased or loss of expression of Loricrin (and optionally Ambra-1). Incertain embodiments, the clear pattern of expression obtained using theantibodies described herein allows rapid and accurate identification ofdecreased or loss of expression of Loricrin (and optionally Ambra-1) byvisual assessment. Thus, certain aspects of the invention relate toimproved methods for determining whether a subject with melanoma has anincreased risk of metastasis. Accordingly, certain aspects of thepresent invention provide inter alia:

-   -   a method for determining whether a subject with melanoma has an        increased risk of metastasis, the method comprising:    -   (i) determining the expression of Loricrin in a tissue sample        obtained from the subject using a monoclonal antibody against        Loricrin as described herein, wherein the tissue sample        comprises tissue overlying a primary melanoma; and    -   (ii) comparing the expression obtained in (i) with a reference        tissue or levels obtained therefrom,        wherein a decrease in the expression of Loricrin in the tissue        sample compared to the reference tissues or levels, or a loss of        expression of Loricrin in the tissue sample, is indicative of an        increased risk of metastasis;    -   use of the antibody against Loricrin as described herein for        determining whether a subject with melanoma has an increased        risk of metastasis;    -   an antibody that competes for binding to Loricrin with the        antibodies as described herein;    -   an antibody that binds to the same epitope as the antibodies as        described herein;    -   a method of labelling Loricrin in a tissue sample overlying a        melanoma, the method comprising:    -   (a) contacting the tissue sample with a monoclonal antibody        against Loricrin as described herein; and    -   (b) visualising the antibody in the tissue sample with a reagent        that generates a detectable signal;    -   a method for determining a treatment regime for a subject        suffering from melanoma, the method comprising:    -   (i) determining the expression of Loricrin in a tissue sample        obtained from the subject using a monoclonal antibody against        Loricrin as described herein, wherein the tissue sample        comprises tissue overlying a primary melanoma; and    -   (ii) comparing the expression obtained in (i) with a reference        tissue or levels obtained therefrom, and    -   (iii) (a) if expression of Loricrin is normal, following a        normal recognized care pathway, or        -   (b) if expression of Loricrin is decreased or lost, treating            the subject with a systemic anti-cancer treatment regime;    -   a method of treating a subject suffering from melanoma, the        method comprising:    -   (i) determining the expression of Loricrin in a tissue sample        obtained from the subject using a monoclonal antibody against        Loricrin as described herein, wherein the tissue sample        comprises tissue overlying a primary melanoma; and    -   (ii) comparing the expression obtained in (i) with a reference        tissue or levels obtained therefrom, and        if there is a decrease in the expression of Loricrin in the        tissue sample compared to the reference tissue or levels, or a        loss of expression of Loricrin, administering a therapeutic        agent to the subject;    -   a method of treating a subject suffering from melanoma, the        method comprising administering a therapeutic agent to the        subject, wherein the subject has been identified as having        decreased or a loss of expression of Loricrin as described        herein;    -   an in vitro assay for predicting an increased risk of metastasis        in a subject suffering from melanoma, the assay comprising:    -   contacting a tissue sample obtained from the subject with an        antibody against Loricrin as described herein, wherein the        tissue sample comprises tissue overlying a primary melanoma and        the presence of Loricrin creates a Loricrin-antibody complex;        and    -   detecting and/or quantifying the Loricrin-antibody complex; and    -   a kit for predicting an increased risk of developing metastasis        of a subject suffering from melanoma, the kit comprising an        antibody against Loricrin as described herein.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Embodiments of the invention will now be described by way of exampleonly, and with reference to the accompanying Figures in which:

FIG. 1 shows identification of recombinant monoclonal fragmentantibodies against human Ambra-1 by ELISA. All HuCAL antibodies havespecificity to Ambra-1 peptide in native/denatured form, with no/littlecross reactivity to control proteins.

FIG. 2 shows identification of recombinant monoclonal fragmentantibodies against human Loricrin by ELISA. All HuCAL antibodies havespecificity to Loricrin antigen in native/denatured form, with no/littlecross reactivity to control proteins, apart from AbD32713.1 which showedbinding to GST and was subsequently omitted from validation on thisbasis.

FIG. 3 shows example photomicrographs of Ambra-1 expression in normalskin tissue NH9813 using Abcam antibody ab69501 and HuCAL antibody33473.

FIG. 4 shows example photomicrographs of Ambra-1 expression in a stage Imelanoma tissue with maintained Ambra-1 expression in epidermisoverlying the tumor, detected using Abcam antibody ab69501 (diluted1/2500) and HuCAL antibody 33473 (10 μg/ml).

FIG. 5 shows example photomicrographs of Ambra-1 expression in a stage Imelanoma tissue with Ambra-1 expression lost in epidermis overlying thetumor, detected using Abcam antibody ab69501 (diluted 1/2500) and HuCALantibody 33473 (10 μg/ml).

FIG. 6 shows example photomicrographs of Loricrin expression in Normalskin Tissue NH9808 using Abcam antibody ab176221 (diluted 1/500) andHuCAL antibody 33047 (20 μg/ml).

FIG. 7 shows example photomicrographs of Loricrin expression in a stageI melanoma tissue with maintained Loricrin expression in epidermisoverlying the tumor, detected using Abcam antibody ab176221 (diluted1/500) and HuCAL antibody 3047 (20 μg/ml).

FIG. 8 shows example photomicrographs of Loricrin expression in a stageI melanoma tissue with Loricrin expression lost in epidermis overlyingthe tumor, detected using Abcam antibody ab176221 (diluted 1/500) andHuCAL antibody 3047 (20 μg/ml).

FIG. 9 shows ELISA results of recombinant monoclonal antibody AbD33473against human Ambra-1.

FIG. 10 shows ELISA results of recombinant monoclonal antibody AbD33047against human Loricrin.

FIG. 11 shows expression of Ambra-1 in AJCC Stage I Melanoma. A)Photomicrograph of AJCC Stage I Melanoma with maintained Ambra-1,detected using Abcam antibody ab69501 (diluted 1/350), or B) HuCALantibody AbD33473 (0.342 μg/ml) using DAB (brown) chromogen. C)Photomicrograph of AJCC Stage I Melanoma with Ambra-1 lost, detectedusing Abcam antibody ab69501 (diluted 1/350), or D) HuCAL antibodyAbD33473 (0.342 μg/ml) using DAB (brown) chromogen. A,B,C,D i.)Magnification approximately 4× or ii.) ×10.

FIG. 12 shows expression of Loricrin in AJCC Stage I Melanoma. A)Photomicrograph of AJCC Stage I Melanoma with maintained Loricrindetected using Abcam antibody ab176322 (diluted 1/2000) using DAB(brown) chromogen, or B) HuCAL antibody AbD33047 (14 μg/ml) using fastred (red) chromogen. C) Photomicrograph of AJCC Stage I Melanoma withLoricrin lost, detected using Abcam antibody ab176322 (diluted 1/2000)using DAB (brown) chromogen, or D) HuCAL antibody AbD33047 (14 μg/ml)using fast red (red) chromogen. A,B,C,D i.) Magnification approximately4× or ii.) ×10.

Sequence listingSEQ ID NO: 1 shows the amino acid sequence of HCDR1 of the anti-Ambra-1 antibody (SYWIH);SEQ ID NO: 2 shows the amino acid sequence of HCDR2 of the anti-Ambra-1 antibody(TIFPSRSYTTYSPSFQG);SEQ ID NO: 3 shows the amino acid sequence of HCDR3 (DTPSTALKSPFDY) of the anti-Ambra-1 antibody;SEQ ID NO: 4 shows the amino acid sequence of LCDR1 of the anti-Ambra-1 antibody(SGSSSNIGYNYVY);SEQ ID NO: 5 shows the amino acid sequence of LCDR2 of the anti-Ambra-1 antibody(ENNKRPS);SEQ ID NO: 6 shows the amino acid sequence of LCDR3 of the anti-Ambra-1 antibody(SSWDSHSNSYV);SEQ ID NO: 7 shows the amino acid sequence of HCFR1 of the anti-Ambra-1 antibody(EVQLVQSGAEVKKPGESLKISCKGSGYSFS);SEQ ID NO: 8 shows the amino acid sequence of HCFR2 of the anti-Ambra-1 antibody(WVRQMPGKGLEWMG);SEQ ID NO: 9 shows the amino acid sequence of HCFR3 of the anti-Ambra-1 antibody(QVTISADKSISTAYLQWSSLKASDTAMYYCAR);SEQ ID NO: 10 shows the amino acid sequence of HCFR4 of the anti-Ambra-1 antibody(WGQGTLVTVSS);SEQ ID NO: 11 shows the amino acid sequence of LCFR1 of the anti-Ambra-1 antibody(DIVLTQPPSVSGAPGQRVTISC);SEQ ID NO: 12 shows the amino acid sequence of LCFR2 of the anti-Ambra-1 antibody(WYQQLPGTAPKLLIY);SEQ ID NO: 13 shows the amino acid sequence of LCFR3 of the anti-Ambra-1 antibody(GVPDRFSGSKSGTSASLAITGLQAEDEADYYC);SEQ ID NO: 14 shows the amino acid sequence of LCFR4 of the anti-Ambra-1 antibody(FGGGTKLTVLGQ);SEQ ID NO: 15 shows the amino acid sequence of the V_(H) domain of the anti-Ambra-1 antibody;EVQLVQSGAEVKKPGESLKISCKGSGYSFSSYWIHWVRQMPGKGLEWMGTIFPSRSYTTYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDTPSTALKSPFDYWGQGTLVTVSSSEQ ID NO: 16 shows the amino acid sequence of the V_(L) domain of the anti-Ambra-1 antibody;DIVLTQPPSVSGAPGQRVTISCSGSSSNIGYNYVYWYQQLPGTAPKLLIYENNKRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCSSWDSHSNSYVFGGGTKLTVLGQSEQ ID NO: 17 shows the amino acid sequence of the Fd chain (V_(H) domain and constant domain)of the Fab region of the anti-Ambra-1 antibody;EVQLVQSGAEVKKPGESLKISCKGSGYSFSSYWIHWVRQMPGKGLEWMGTIFPSRSYTTYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDTPSTALKSPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSEQ ID NO: 18 shows the amino acid sequence of the light chain (V_(L) domain and constantdomain) of the Fab region of the anti-Ambra-1 antibody;DIVLTQPPSVSGAPGQRVTISCSGSSSNIGYNYVYWYQQLPGTAPKLLIYENNKRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCSSWDSHSNSYVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEASEQ ID NO: 19 shows the nucleic acid sequence encoding the Fd chain of the Fab region andtags (alkaline phosphatase dimerization domain sequence (AP), FLAG tag (DYKDDDDK) andHis6 tag of the anti-Ambra-1 antibody;GAAGTGCAATTGGTGCAGAGCGGTGCGGAAGTGAAAAAACCGGGCGAAAGCCTGAAAATTAGCTGCAAAGGCTCCGGATATAGCTTCTCTTCTTACTGGATCCATTGGGTGCGCCAGATGCCGGGCAAAGGTCTCGAGTGGATGGGCACTATCTTCCCGTCTCGTAGCTACACCACTTATAGCCCGAGCTTTCAGGGCCAGGTGACCATTAGCGCGGATAAAAGCATCAGCACCGCGTATCTGCAATGGAGCAGCCTGAAAGCGAGCGATACCGCGATGTATTATTGCGCGCGTGACACTCCGTCTACTGCTCTGAAATCTCCGTTCGATTACTGGGGCCAAGGCACCCTGGTGACTGTTAGCTCAGCGTCGACCAAAGGCCCGAGCGTGTTTCCGCTGGCCCCGAGCAGCAAAAGCACCAGCGGCGGCACCGCCGCACTGGGCTGCCTGGTGAAAGATTATTTCCCGGAACCAGTGACCGTGAGCTGGAACAGCGGTGCCCTGACCAGCGGCGTGCATACCTTTCCGGCGGTGCTGCAAAGCAGCGGCCTGTATAGCCTGAGCAGCGTTGTGACCGTGCCGAGCAGCAGCCTGGGCACCCAGACCTATATTTGCAACGTCAACCATAAACCGAGCAACACCAAAGTCGATAAAAAAGTCGAACCGAAAAGCGAATTCAAGGCTGAAATGCCTGTTCTGGAAAACCGGGCTGCTCAGGGCGATATTACTACACCCGGCGGTGCTCGCCGTTTAACGGGTGATCAGACTGCCGCTCTGCGTGATTCTCTTAGCGATAAACCTGCAAAAAATATTATTTTGCTGATTGGCGATGGGATGGGGGACTCGGAAATTACTGCCGCACGTAATTATGCCGAAGGTGCGGGCGGCTTTTTTAAAGGTATAGATGCCTTACCGCTTACCGGGCAATACACTCACTATGCGCTGAATAGAAAAACCGGCAAACCGGACTACGTCACCAGCTCGGCTGCATCAGCAACCGCCTGGTCAACCGGTGTCAAAACCTATAACGGCGCGCTGGGCGTCGATATTCACGAAAAAGATCACCCAACGATTCTGGAAATGGCAAAAGCCGCAGGTCTGGCGACCGGTAACGTTTCTACCGCAGAGTTGCAGGATGCCACGCCCGCTGCGCTGGTGGCACATGTGACCTCGCGCAAATGCTACGGTCCGAGCGCGACCAGTGAAAAATGTCCGGGTAACGCTCTGGAAAAAGGCGGAAAAGGATCGATTACCGAACAGCTGCTTAACGCTCGTGCCGACGTTACGCTTGGCGGCGGCGCAAAAACCTTTGCTGAAACGGCAACCGCTGGTGAATGGCAGGGAAAAACGCTGCGTGAACAGGCACAGGCGCGTGGTTATCAGTTGGTGAGCGATGCTGCCTCACTGAACTCGGTGACGGAAGCGAATCAGCAAAAACCCCTGCTTGGCCTGTTTGCTGACGGCAATATGCCAGTGCGCTGGCTAGGACCGAAAGCAACGTACCATGGCAATATCGATAAGCCCGCAGTCACCTGTACGCCAAATCCGCAACGTAATGACAGTGTACCAACCCTGGCGCAGATGACCGACAAAGCCATTGAATTGTTGAGTAAAAATGAGAAAGGCTTTTTCCTGCAAGTTGAAGGTGCGTCAATCGATAAACAGGATCATGCTGCGAATCCTTGTGGGCAAATTGGCGAGACGGTCGATCTCGATGAAGCCGTACAACGGGCGCTGGAGTTCGCTAAAAAGGAGGGTAACACGCTGGTCATAGTCACCGCTGATCACGCCCACGCCAGCCAGATTGTTGCGCCGGATACCAAAGCTCCGGGCCTCACCCAGGCGCTAAATACCAAAGATGGCGCAGTGATGGTGATGAGTTACGGGAACTCCGAAGAGGATTCACAAGAACATACCGGCAGTCAGTTGCGTATTGCGGCGTATGGCCCGCATGCCGCCAATGTTGTTGGACTGACCGACCAGACCGATCTCTTCTACACCATGAAAGCCGCTCTGGGGCTGAAAGGCGCGCCGGACTATAAAGATGACGATGACAAAGGCGCGCCGCACCATCATCACCATCACSEQ ID NO: 20 shows the nucleic acid sequence encoding the light chain of the Fab region ofthe anti-Ambra-1 antibody;GATATCGTGCTGACCCAGCCGCCGAGCGTGAGCGGTGCACCGGGCCAGCGCGTGACCATTAGCTGTAGCGGCAGCAGCAGCAACATTGGTTACAACTACGTGTACTGGTACCAGCAGCTGCCGGGCACGGCGCCGAAACTGCTGATCTACGAAAACAACAAACGCCCGAGCGGCGTGCCGGATCGCTTTAGCGGATCCAAAAGCGGCACCAGCGCCAGCCTGGCGATTACCGGCCTGCAAGCAGAAGACGAAGCGGATTATTACTGCTCTTCTTGGGACTCTCATTCTAACTCTTACGTGTTTGGCGGCGGCACGAAGTTAACCGTTCTTGGCCAGCCGAAAGCCGCCCCAAGCGTGACCCTGTTTCCGCCGAGCAGCGAAGAACTGCAAGCCAACAAAGCCACCCTGGTTTGCCTGATCAGCGATTTTTATCCGGGTGCCGTGACCGTGGCCTGGAAAGCCGATAGCAGCCCGGTGAAAGCCGGCGTGGAAACCACCACCCCGAGCAAACAGAGCAACAACAAATATGCCGCCAGCAGCTATCTGAGCCTGACCCCGGAACAGTGGAAAAGCCATCGCAGCTATAGTTGTCAAGTGACCCATGAAGGCAGCACCGTGGAAAAAACCGTGGCCCCGACCGAGGCCSEQ ID NO: 21 shows the amino acid sequence of human Ambra-1;MKVVPEKNAVRILWGRERGARAMGAQRLLQELVEDKTRWMKWEGKRVELPDSPRSTFLLAFSPDRTLLASTHVNHNIYITEVKTGKCVHSLIGHRRTPWCVTFHPTISGLIASGCLDGEVRIWDLHGGSESWFTDSNNAIASLAFHPTAQLLLIATANEIHFWDWSRREPFAVVKTASEMERVRLVRFDPLGHYLLTAIVNPSNQQGDDEPEIPIDGTELSHYRQRALLQSQPVRRTPLLHNFLHMLSSRSSGIQVGEQSTVQDSATPSPPPPPPQPSTERPRTSAYIRLRQRVSYPTAECCQHLGILCLCSRCSGTRVPSLLPHQDSVPPASARATTPSFSFVQTEPFHPPEQASSTQQDQGLLNRPSAFSTVQSSTAGNTLRNLSLGPTRRSLGGPLSSHPSRYHREIAPGLTGSEWTRTVLSLNSRSEAESMPPPRTSASSVSLLSVLRQQEGGSQASVYTSATEGRGFPASGLATESDGGNGSSQNNSGSIRHELQCDLRRFFLEYDRLQELDQSLSGEAPQTQQAQEMLNNNIESERPGPSHQPTPHSSENNSNLSRGHLNRCRACHNLLTFNNDTLRWERTTPNYSSGEASSSWQVPSSFESVPSSGSQLPPLERTEGQTPSSSRLELSSSASPQEERTVGVAFNQETGHWERIYTQSSRSGTVSQEALHQDMPEESSEEDSLRRRLLESSLISLSRYDGAGSREHPIYPDPARLSPAAYYAQRMIQYLSRRDSIRQRSMRYQQNRLRSSTSSSSSDNQGPSVEGTDLEFEDFEDNGDRSRHRAPRNARMSAPSLGRFVPRRFLLPEYLPYAGIFHERGQPGLATHSSVNRVLAGAVIGDGQSAVASNIANTTYRLQWWDFTKFDLPEISNASVNVLVQNCKIYNDASCDISADGQLLAAFIPSSQRGFPDEGILAVYSLAPHNLGEMLYTKRFGPNAISVSLSPMGRYVMVGLASRRILLHPSTEHMVAQVFRLQQAHGGETSMRRVFNVLYPMPADQRRHVSINSARWLPEPGLGLAYGTNKGDLVICRPEALNSGVEYYWDQLNETVFTVHSNSRSSERPGTSRATWRTDRDMGLMNAIGLQPRNPATSVTSQGTQTLALQLQNAETQTEREVPEPGTAASGPGEGEGSEYGASGEDALSRIQRLMAEGGMTAVVQREQSTTMASMGGFGNNIIVSHRIHRSSQTGTEPGAAHTSSPQPSTSRGLLPEAGQLAERGLSPRTASWDQPGTPGREPTQPTLPSSSPVPIPVSLPSAEGPTLHCELTNNNHLLDGGSSRGDAAGPRGEPRNRSEQ ID NO: 22 shows the amino acid sequence of HCDR1 of the anti-Loricrin antibody (DYYIH);SEQ ID NO: 23 shows the amino acid sequence of HCDR2 of the anti-Loricrin antibody(VISPNSGKTNYAQKFQG);SEQ ID NO: 24 shows the amino acid sequence of HCDR3 of the anti-Loricrin antibody(DLYPDSSAFDI);SEQ ID NO: 25 shows the amino acid sequence of LCDR1 of the anti-Loricrin antibody(SGDNLGDKYAH);SEQ ID NO: 26 shows the amino acid sequence of LCDR2 of the anti-Loricrin antibody(DDNERPS);SEQ ID NO: 27 shows the amino acid sequence of LCDR3 of the anti-Loricrin antibody(QSYDSGNRV);SEQ ID NO: 28 shows the amino acid sequence of HCFR1 of the anti-Loricrin antibody(QVQLVQSGAEVKKPGASVKVSCKASGYTFN);SEQ ID NO: 29 shows the amino acid sequence of HCFR2 of the anti-Loricrin antibody(WVRQAPGQGLEWMG);SEQ ID NO: 30 shows the amino acid sequence of HCFR3 of the anti-Loricrin antibody(RVTMTRDTSISTAYMELSRLRSEDTAVYYCAR);SEQ ID NO: 31 shows the amino acid sequence of HCFR4 of the anti-Loricrin antibody(WGQGTLVTVSS);SEQ ID NO: 32 shows the amino acid sequence of LCFR1 of the anti-Loricrin antibody(DIELTQPPSVSVSPGQTASITC);SEQ ID NO: 33 shows the amino acid sequence of LCFR2 of the anti-Loricrin antibody(WYQQKPGQAPVLVIY);SEQ ID NO: 34 shows the amino acid sequence of LCFR3 of the anti-Loricrin antibody(GIPERFSGSNSGNTATLTISGTQAEDEADYYC);SEQ ID NO: 35 shows the amino acid sequence of LCFR4 of the anti-Loricrin antibody(FGGGTKLTVLGQ);SEQ ID NO: 36 shows the amino acid sequence of the V_(H) domain of the anti-Loricrin antibody;QVQLVQSGAEVKKPGASVKVSCKASGYTFNDYYIHWVRQAPGQGLEWMGVISPNSGKTNYAQKFQGRVTMTRDTSISTAYMELSRLRSEDTAVYYCARDLYPDSSAFDIWGQGTLVTVSSSEQ ID NO: 37 shows the amino acid sequence of the V_(L) domain of the anti-Loricrin antibody;DIELTQPPSVSVSPGQTASITCSGDNLGDKYAHWYQQKPGQAPVLVIYDDNERPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQSYDSGNRVFGGGTKLTVLGQSEQ ID NO: 38 shows the amino acid sequence of the Fd chain (V_(H) domain and constant domain)of the Fab region of the anti-Loricrin antibody;QVQLVQSGAEVKKPGASVKVSCKASGYTFNDYYIHWVRQAPGQGLEWMGVISPNSGKTNYAQKFQGRVTMTRDTSISTAYMELSRLRSEDTAVYYCARDLYPDSSAFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSEQ ID NO: 39 shows the amino acid sequence of the light chain (V_(L) domain and constantdomain) of the Fab region of the anti-Loricrin antibody;DIELTQPPSVSVSPGQTASITCSGDNLGDKYAHWYQQKPGQAPVLVIYDDNERPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQSYDSGNRVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEASEQ ID NO: 40 shows the nucleic acid sequence encoding the Fd chain of the Fab region andtags (alkaline phosphatase dimerization domain sequence (AP), FLAG tag (DYKDDDDK) andHis6 tag (HHHHHH)) of the anti-Loricrin antibody;CAGGTGCAATTGGTGCAGAGCGGTGCGGAAGTGAAAAAACCGGGTGCCAGCGTGAAAGTTAGCTGCAAAGCGTCCGGATATACCTTCAACGACTACTACATCCATTGGGTGCGCCAGGCCCCGGGCCAGGGCCTCGAGTGGATGGGCGTTATCTCTCCGAACTCTGGCAAAACGAACTACGCGCAGAAATTTCAGGGCCGGGTGACCATGACCCGTGATACCAGCATTAGCACCGCGTATATGGAACTGAGCCGTCTGCGTAGCGAAGATACGGCCGTGTATTATTGCGCGCGTGACCTGTACCCGGACTCTTCTGCTTTCGATATCTGGGGCCAAGGCACCCTGGTGACTGTTAGCTCAGCGTCGACCAAAGGCCCGAGCGTGTTTCCGCTGGCCCCGAGCAGCAAAAGCACCAGCGGCGGCACCGCCGCACTGGGCTGCCTGGTGAAAGATTATTTCCCGGAACCAGTGACCGTGAGCTGGAACAGCGGTGCCCTGACCAGCGGCGTGCATACCTTTCCGGCGGTGCTGCAAAGCAGCGGCCTGTATAGCCTGAGCAGCGTTGTGACCGTGCCGAGCAGCAGCCTGGGCACCCAGACCTATATTTGCAACGTCAACCATAAACCGAGCAACACCAAAGTCGATAAAAAAGTCGAACCGAAAAGCGAATTCAAGGCTGAAATGCCTGTTCTGGAAAACCGGGCTGCTCAGGGCGATATTACTACACCCGGCGGTGCTCGCCGTTTAACGGGTGATCAGACTGCCGCTCTGCGTGATTCTCTTAGCGATAAACCTGCAAAAAATATTATTTTGCTGATTGGCGATGGGATGGGGGACTCGGAAATTACTGCCGCACGTAATTATGCCGAAGGTGCGGGCGGCTTTTTTAAAGGTATAGATGCCTTACCGCTTACCGGGCAATACACTCACTATGCGCTGAATAGAAAAACCGGCAAACCGGACTACGTCACCAGCTCGGCTGCATCAGCAACCGCCTGGTCAACCGGTGTCAAAACCTATAACGGCGCGCTGGGCGTCGATATTCACGAAAAAGATCACCCAACGATTCTGGAAATGGCAAAAGCCGCAGGTCTGGCGACCGGTAACGTTTCTACCGCAGAGTTGCAGGATGCCACGCCCGCTGCGCTGGTGGCACATGTGACCTCGCGCAAATGCTACGGTCCGAGCGCGACCAGTGAAAAATGTCCGGGTAACGCTCTGGAAAAAGGCGGAAAAGGATCGATTACCGAACAGCTGCTTAACGCTCGTGCCGACGTTACGCTTGGCGGCGGCGCAAAAACCTTTGCTGAAACGGCAACCGCTGGTGAATGGCAGGGAAAAACGCTGCGTGAACAGGCACAGGCGCGTGGTTATCAGTTGGTGAGCGATGCTGCCTCACTGAACTCGGTGACGGAAGCGAATCAGCAAAAACCCCTGCTTGGCCTGTTTGCTGACGGCAATATGCCAGTGCGCTGGCTAGGACCGAAAGCAACGTACCATGGCAATATCGATAAGCCCGCAGTCACCTGTACGCCAAATCCGCAACGTAATGACAGTGTACCAACCCTGGCGCAGATGACCGACAAAGCCATTGAATTGTTGAGTAAAAATGAGAAAGGCTTTTTCCTGCAAGTTGAAGGTGCGTCAATCGATAAACAGGATCATGCTGCGAATCCTTGTGGGCAAATTGGCGAGACGGTCGATCTCGATGAAGCCGTACAACGGGCGCTGGAGTTCGCTAAAAAGGAGGGTAACACGCTGGTCATAGTCACCGCTGATCACGCCCACGCCAGCCAGATTGTTGCGCCGGATACCAAAGCTCCGGGCCTCACCCAGGCGCTAAATACCAAAGATGGCGCAGTGATGGTGATGAGTTACGGGAACTCCGAAGAGGATTCACAAGAACATACCGGCAGTCAGTTGCGTATTGCGGCGTATGGCCCGCATGCCGCCAATGTTGTTGGACTGACCGACCAGACCGATCTCTTCTACACCATGAAAGCCGCTCTGGGGCTGAAAGGCGCGCCGGACTATAAAGATGACGATGACAAAGGCGCGCCGCACCATCATCACCATCACSEQ ID NO: 41 shows the nucleic acid sequence encoding the light chain of the Fab region ofthe anti-Loricrin antibody;GATATCGAACTGACCCAGCCGCCGAGCGTGAGCGTGAGCCCGGGCCAGACCGCGAGCATTACCTGTAGCGGCGATAACCTGGGTGACAAATACGCTCATTGGTACCAGCAGAAACCGGGCCAGGCGCCGGTGCTGGTGATCTACGACGACAACGAACGTCCGAGCGGCATCCCGGAACGTTTTAGCGGATCCAACAGCGGCAACACCGCGACCCTGACCATTAGCGGCACCCAGGCGGAAGACGAAGCGGATTATTACTGCCAGTCTTACGACTCTGGTAACCGTGTGTTTGGCGGCGGCACGAAGTTAACCGTTCTTGGCCAGCCGAAAGCCGCCCCAAGCGTGACCCTGTTTCCGCCGAGCAGCGAAGAACTGCAAGCCAACAAAGCCACCCTGGTTTGCCTGATCAGCGATTTTTATCCGGGTGCCGTGACCGTGGCCTGGAAAGCCGATAGCAGCCCGGTGAAAGCCGGCGTGGAAACCACCACCCCGAGCAAACAGAGCAACAACAAATATGCCGCCAGCAGCTATCTGAGCCTGACCCCGGAACAGTGGAAAAGCCATCGCAGCTATAGTTGTCAAGTGACCCATGAAGGCAGCACCGTGGAAAAAACCGTGGCCCCGACCGAGGCC SEQ ID NO: 42 shows the amino acid sequence of human Loricrin;MSYQKKQPTPQPPVDCVKTSGGGGGGGGSGGGGCGFFGGGGSGGGSSGSGCGYSGGGGYSGGGCGGGSSGGGGGGGIGGCGGGSGGSVKYSGGGGSSGGGSGCFSSGGGGSGCFSSGGGGSSGGGSGCFSSGGGGSSGGGSGCFSSGGGGFSGQAVQCQSYGGVSSGGSSGGGSGCFSSGGGGGSVCGYSGGGSGCGGGSSGGSGSGYVSSQQVTQTSCAPQPSYGGGSSGGGGSGGSGCFSSGGGGGSSGCGGGSSGIGSGCIISGGGSVCGGGSSGGGGGGSSVGGSGSGKGVPICHQTQQKQAPTWPSKSEQ ID NO: 43 shows the amino acid sequence of an exemplary epitope tag(DYKDDDDK)SEQ ID NO: 44 shows the amino acid sequence of an exemplary epitope tag(GKPIPNPLLGLDST)SEQ ID NO: 45 shows the amino acid sequence of an exemplary epitope tag(WSHPQFEK)SEQ ID NO: 46 shows the amino acid sequence of the peptide to which anti-Ambra-1 antibodieswere raised (CGGSSRGDAAGPRGEPRNR)SEQ ID NO: 47 shows the amino acid sequence of the His6 tag (HHHHHH)SEQ ID NO: 48 shows the Ambra-1 C-terminal sequence used for replacement analysis inepitope mapping (VSLPSAEGPTLHCELTNNNHLLDGGSSRGDAAGPRGEPRNR)SEQ ID NO: 49 shows the REPNET epitope of the Anti-Ambra-1 antibodies(DGGSSRGDAAGPRGEPRNR)SEQ ID NO: 50 shows the LIN epitope of the Anti-Ambra-1 antibodies (HLLDGGSSR)SEQ ID NO: 51 shows the LOOP epitope of the Anti-Ambra-1 antibodies (NHLLDGGSSR)SEQ ID NO: 52 shows a core epitope of the Anti-Ambra-1 antibodies (EPRN)SEQ ID NO: 53 shows a core epitope of the Anti-Ambra-1 antibodies (EPR)SEQ ID NO: 54 shows an epitope of the Anti- Loricrin antibodies(SYQKKQPTPGPPVDCVKTS)SEQ ID NO: 55 shows an epitope of the Anti- Loricrin antibodies(GGGGIGGPGGGSGGSVKYS)SEQ ID NO: 56 shows an epitope of the Anti- Loricrin antibodies(GSSGGGSGCFSSGGG)SEQ ID NO: 57 shows an epitope of the Anti- Loricrin antibodies(GIGSGCIISGGGSVCGGGS)SEQ ID NO: 58 shows an epitope of the Anti- Loricrin antibodies(GSSGGGGGGSSVGGSGSGK)SEQ ID NO: 59 shows an epitope of the Anti- Loricrin antibodies (GVCICHQTQQK)SEQ ID NO: 60 shows a core epitope of the Anti- Loricrin antibodies (VKYS)SEQ ID NO: 61 shows a core epitope of the Anti- Loricrin antibodies (CFS)

The practice of embodiments of the present invention employs, unlessotherwise indicated, conventional techniques of chemistry, molecularbiology, pharmaceutical formulation, pharmacology and medicine, whichare within the skill of those working in the art.

Most general chemistry techniques can be found in ComprehensiveHeterocyclic Chemistry IF (Katritzky et al., 1996, published by PergamonPress); Comprehensive Organic Functional Group Transformations(Katritzky et al., 1995, published by Pergamon Press); ComprehensiveOrganic Synthesis (Trost et al., 1991, published by Pergamon);Heterocyclic Chemistry (Joule et al. published by Chapman & Hall);Protective Groups in Organic Synthesis (Greene et al., 1999, publishedby Wiley-Interscience); and Protecting Groups (Kocienski et al., 1994).

Most general molecular biology techniques can be found in Sambrook etal, Molecular Cloning, A Laboratory Manual (2001) Cold Harbor-LaboratoryPress, Cold Spring Harbor, N.Y. or Ausubel et al., Current Protocols inMolecular Biology (1990) published by John Wiley and Sons, N.Y.

Most general pharmaceutical formulation techniques can be found inPharmaceutical Preformulation and Formulation (2^(nd) Edition edited byMark Gibson) and Pharmaceutical Excipients: Properties, Functionalityand Applications in Research and Industry (edited by Otilia M Y Koo,published by Wiley).

Most general pharmacological techniques can be found in A Textbook ofClinical Pharmacology and Therapeutics (5^(th) Edition published byArnold Hodder).

Most general techniques on the prescribing, dispensing and administeringof medicines can be found in the British National Formulary 72(published jointly by BMJ Publishing Group Ltd and Royal PharmaceuticalSociety).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3^(rd)ed., Academic Press; and the Oxford University Press, provide a personskilled in the art with a general dictionary of many of the terms usedin this disclosure. For chemical terms, the skilled person may refer tothe International Union of Pure and Applied Chemistry (IUPAC).

Units, prefixes and symbols are denoted in their Système Internationald'Unités (SI) accepted form. Numeric ranges are inclusive of the numbersdefining the range.

Methods for Determining Risk of Progression to Metastatic Melanoma

The invention provides a method for determining whether a subject withmelanoma has an increased risk of metastasis. Typically, the method isin vitro. The subject may be a human or an animal suffering frommelanoma. In some embodiments, the subject is a horse, cat or dog.Typically, the subject is a human. In some embodiments, the subject hasalready been diagnosed as having melanoma.

As used herein, the term “metastasis” refers to the recurrence ordisease progression that may occur locally (such as local recurrence andin transit disease), regionally (such as nodal micro-metastasis ormacro-metastasis), or distally (such as brain, lung and other tissues).In some embodiments, the term “metastasis” is used to refer tometastatic disease following a primary melanoma. Typically, metastasisoriginating from a primary melanoma may spread to the lungs and/or brainof the subject as well as other locations.

The stage of a melanoma is a description of how widespread it is. Thisincludes its thickness in the skin, whether it has spread to nearbylymph nodes or any other organs, and certain other factors. The stage isbased on the results of the physical exam, biopsies, and any imagingtests (CT or MRI scan, etc.) or other tests that have been done. Suchtests will be known to those skilled in the art. The system most oftenused to stage melanoma is the American Joint Commission on Cancer (AJCC)TNM system. The table below describes the features identifying eachstage.

Stage 1 1a Tumour <1.00 mm without ulceration; no lymph nodeinvolvement, no distant metastases. 1b Tumour <1.00 mm with ulcerationor Clark level IV or V tumour 1.01-2.0 mm without ulceration; no lymphnode involvement; no distant metastases. Stage 2 2a Tumour 1.01-2.0 mmwith ulceration; tumour 2.01-4.0 mm without ulceration; no lymph nodeinvolvement; no distant metastases. 2b Tumour 2.01-4 mm with ulceration.2b Tumour >4.0 mm without ulceration; no lymph node involvement; nodistant metastases. 2c Tumour >4.0 mm with ulceration; no nodalinvolvement; no distant metastases. Stage 3 3a Tumour of any thicknesswithout ulceration with 1 positive lymph node and micro-metastasis ormacro-metastasis. 3b Tumour of any thickness without ulceration with 2-3positive lymph nodes and micro-metastasis or macro-metastasis. 3c Tumourof any thickness and macro-metastasis OR in-transit met(s)/satellite(s)without metastatic lymph nodes, OR 4 or more metastatic lymph nodes,matted nodes or combinations of in-transit met(s)/satellite(s), ORulcerated melanoma and metastatic lymph node(s). Stage 4 4 Tumour of anythickness with any nodes and any metastases

In some embodiments, the subject is suffering from AJCC stage 1, stage2, stage 3 or stage melanoma. In some embodiments, the subject issuffering from AJCC stage 1a, stage 1b, stage 2a, stage 2b or stage 2cmelanoma. In some embodiments, the subject is suffering from AJCC stage1a or stage 1b melanoma. In some embodiments, the methods furthercomprise staging a primary tumour present in a tissue sample obtainedfrom a subject in accordance with AJCC staging. In some embodiments thepatient has ulcerated melanoma.

The method for determining whether a subject with melanoma has anincreased risk of metastasis comprises determining the expression ofLoricrin in a tissue sample obtained from the subject.

The tissue sample comprises tissue overlying a primary melanoma. As usedherein, the term “primary melanoma” refers to a malignant tumour on theskin at the site of origin, regardless of thickness, in patients withoutclinical or histologic evidence of regional or distant metastaticdisease. The wording “tissue overlying a primary melanoma”, refers toepidermal tissue situated between a primary melanoma and the surface ofthe skin.

In some embodiments, the tissue sample comprises at least a portion ofthe peri-tumoral epidermis overlying the primary melanoma. In someembodiments, the tissue sample further comprises a portion of normaltissue adjacent to the primary melanoma. The term “normal tissue”includes, for example, “normal epidermis” which is healthy (i.e.disease-free). In some embodiments, the normal tissue is epidermis thatlies adjacent to the primary melanoma, for example, within a cuff ofnormal skin taken with the primary melanoma sample. The term“peri-tumoral epidermis” refers to epidermal tissue overlying orsituated around a tumor.

In some embodiments, the method comprises determining the expressionlevels of Loricrin in the epidermis. Keratinocytes are cells whichconstitute about 90% of the epidermis. Thus, in some embodiments, thetissue sample comprises keratinocytes overlying the primary melanoma.Typically, the subject may be identified as being at increased risk ofmetastasis, wherein said identification comprises determining that thesubject has a decrease or loss of expression of Loricrin inkeratinocytes overlying the primary melanoma of the subject.

In some embodiments, the tissue sample has previously been obtained fromthe subject such that the sampling itself does not form a part of themethods of the invention. The sample may have been obtained immediatelyprior to the method, or hours, days or weeks prior to the method. Inother embodiments, a method of the invention may additionally comprisethe step of obtaining the tissue sample from the subject.

The expression of Loricrin is determined using a monoclonal antibodyagainst Loricrin as described herein. Typically, the expression ofLoricrin is determined by contacting the tissue with the antibody anddetecting the presence of the bound antibody. For example, presence ofthe bound antibody may be detected by visualising the antibody in thetissue sample with a reagent that generates a detectable signal (e.g. adetection moiety as described herein).

In some embodiments, the method comprises contacting the tissue with theantibody under conditions permissive for binding of the anti-Loricrinantibody to Loricrin and detecting whether a complex is formed betweenthe anti-Loricrin antibody and Loricrin. Such methods may be in vitro orin vivo methods. Typically, the presence of the bound antibody isdetected by immunoassays such as immunohistochemistry (IHC),immunofluorescence (IF), immunoblotting, flow cytometry (e.g., FACS™) orenzyme-linked immunosorbent assay (ELISA).

The method for determining whether a subject with melanoma has anincreased risk of metastasis further comprises comparing the expressionof Loricrin determined for the tissue sample obtained from the subjectwith a reference tissue or levels obtained therefrom.

In some embodiments, the reference comprises levels of Loricrinexpression that are characteristic of normal tissue. Typically,reference levels of Loricrin may be obtained by determining theexpression of Loricrin in a reference tissue. In some embodiments, theexpression levels of Loricrin in a reference tissue are determined byvisual or automated assessment. In some embodiments, reference levels ofLoricrin expression that are characteristic of normal tissue areobtained by determining expression levels in tissue samples obtainedfrom one or more (e.g. a cohort) of healthy subjects.

In some embodiments, the reference tissue comprises normal tissue. Insome embodiments, the normal tissue comprises epidermis from a sitewhich does not include a primary melanoma. In some embodiments, thereference tissue (or levels obtained therefrom) is an internal reference(i.e. obtained from the subject). In some embodiments, the referencetissue is normal tissue obtained from a site adjacent to the primarymelanoma. In other embodiments, the reference tissue is obtained from asite of the subject which is remote from the primary melanoma. Thus, insome embodiments, the reference level is the level of expression ofLoricrin in normal tissue. The reference tissue may be taken from normalepidermis and the reference level is a level of expression in thekeratinocytes of the normal epidermis. The expression of Loricrin in thereference tissue, for example, to generate reference levels, can bedetermined using the methods described herein.

Typically, the tissue sample may be a biopsy, or a section thereof,obtained from the subject. A tissue sample, such as a biopsy, can beobtained through a variety of sampling methods known to those skilled inthe art, including a punch biopsy, shave biopsy, wide local excision andother means. Aptly, the tumour sample is taken from a surgical site fromwhich the primary melanoma has been excised from a subject.

Typically, the tissue sample may be frozen, fresh, fixed (e.g. formalinfixed), centrifuged, and/or embedded (e.g. paraffin embedded), etc. Thetissue sample may be or have been subjected to a variety of well-knownpost-collection preparative and storage techniques (e.g., nucleic acidand/or protein extraction, fixation, storage, freezing, ultrafiltration,concentration, evaporation, centrifugation, etc.) prior to assessing theamount of the Loricrin in the sample. Likewise, biopsies may also besubjected to post-collection preparative and storage techniques, e.g.,fixation. A tissue sample, or a section thereof, may be mounted on asolid support, such as a slide.

In the method for determining whether a subject with melanoma has anincreased risk of metastasis, a decrease in the expression of Loricrinin the tissue sample compared to the reference tissue or levels, or aloss of expression of Loricrin in the tissue sample, is indicative of anincreased risk of metastasis.

For example, a decrease or loss in the expression of Loricrin may be alevel of expression of Loricrin less than about 75% of the respectivereference level.

In some embodiments, a decrease in expression of Loricrin is a level ofexpression of Loricrin from about 25% to about 75% of the respectivereference level. In some embodiments, a loss of expression of Loricrinis a level of expression of Loricrin less than about 25% of thereference level of the relevant protein. Normal expression is understoodto mean that the expression of Loricrin is greater than about 75% of thereference level. Thus, in some embodiments, the expression level ofLoricrin in the tissue sample is from about 25% to about 75% of thereference level. In some embodiments, the expression level of Loricrinis no greater than 75%, no greater than 70%, no greater than 60%, nogreater than 50%, no greater than 40% or no greater than 30% of thereference level. In some embodiments, there is substantially noexpression of Loricrin in the tissue sample. In certain embodiments, theexpression of Loricrin is less than 25%.

The term “comparing” and “compare” may refer to a comparison ofcorresponding parameters or levels, e.g., an absolute amount is comparedto an absolute reference amount while a concentration is compared to areference concentration or a signal intensity signal obtained from thetissue sample is compared to the same type of signal intensity obtainedfrom the reference.

The comparison may be carried out manually, for example by visualassessment, or it may be automated (e.g. using an automated scanner orcomputer-assisted). Thus, the comparison may be carried out by acomputing device.

In certain embodiments, the expression of Loricrin is scored on thebasis of the intensity and/or proportion of positive cells in the tissuesample. Scoring methods have been described and are well known to one ofordinary skill in the art.

In one embodiment, an intensity score may be defined as follows: 0=noappreciable staining in the cells, 1=faint/barely perceptible partialstaining in the cytoplasm and/or nucleus of the cells, 2=weak tomoderate staining of the cytoplasm and/or nucleus of the cells, and3=strong staining of the cytoplasm and/or nucleus of the cells. Aproportion score may be defined as follows: 0=less than 5%, 1=from 5% to25%, 2=from 26% to 50%, 3=from 51% to 75%, and 4=more than 75%. A totalscore may be calculated by multiplying the intensity score and theproportion score, producing a total range of 0 to 12. In certainembodiments, scores of 0 to 3 may be indicative of decrease or loss ofexpression. In certain embodiments, scores of 4 to 12 may be indicativeof an increase in expression.

In another embodiment, a H-score may be calculated (McCarty et al.,Cancer Res. 1986 46(8 Supl):4244s-4248s). An intensity score may bedefined as follows:

1 = faint/barely perceptible partial (e.g. weak) staining in the cells 2= moderate staining of the cells; 3 = strong staining of the cells.

The H score combines components of staining intensity with thepercentage of positive cells. It has a value between 0 and 300 and isdefined as:

1*(percentage of cells staining at 1+ intensity);+2*(percentage of cells staining at 2+ intensity);+3*(percentage of cells staining at 3+ intensity);=H score.

A H-score of about 100, 110, 120, 130, 140, 150 or above may indicateincreased expression of Loricrin. Conversely, a H-score of less thanabout 150, 140, 130, 120, 110 or 100 may indicate decreased expressionof Loricrin.

In certain embodiments, “weak”, “moderate” or “strong” staining of thecells is relative to levels of Loricrin characteristic of the referenceor normal tissue

In certain embodiments, the method of comparing the expression ofLoricrin comprises outputting, optionally on a computer, (i) anindication of the expression levels of Loricrin and (ii) this indicateswhether the subject has an increased risk of metastasis.

In certain embodiments, an increased risk of metastasis means a 7-yearmetastasis-free (also known as “disease-free”) survival rate of lessthan 50%, for example less than 40%, for example less than 30%, forexample less than 20%, for example less than 10%, for example less than5%.

The methods described herein allow subjects with melanoma to bestratified into those more likely to develop metastasis and those lesslikely to develop metastasis. Advantageously, the methods of theinvention help to identify which subjects with melanoma are most likelyto benefit from treatment with a therapeutic agent. Typically, methodsof the invention enable treatment with a therapeutic agent for a subjectwho would otherwise not have been eligible for treatment with atherapeutic agent.

Antibodies Against Loricrin

Antibodies against Loricrin are used to determine the expression ofLoricrin in a tissue sample obtained from the subject.

Antibodies against Loricrin include any monoclonal antibodies, includingchimeric antibodies, humanized antibodies, bi-specific antibodies anddomains and fragments of monoclonal antibodies including Fab, Fab′,F(ab′)2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, andmultimers thereof. Monoclonal antibodies can be fragmented usingconventional techniques. Monoclonal antibodies may be from any animalorigin, including birds and mammals (e.g., human, murine, donkey, sheep,rabbit, goat, guinea pig, camel, horse, or chicken), transgenic animals,or from recombinant sources. Typically, the monoclonal antibodiesagainst Loricrin are fully human.

Monoclonal antibodies may be prepared using any methods known to thoseskilled in the art, including by recombination. Aptly, the antibodies ofthe invention exclude polyclonal antibodies such as research-gradeantibodies.

Typically, the antibody against Loricrin is isolated. An “isolated”antibody is an antibody that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would interferewith diagnostic or therapeutic uses for the antibody, and may includeenzymes, hormones, and other proteinaceous or non-proteinaceous solutes.In certain embodiments, the antibody will be purified (1) to greaterthan 95% by weight of antibody as determined by the Lowry method, andmost preferably more than 99% by weight, (2) to a degree sufficient toobtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator, or (3) to homogeneity bySDS-PAGE under reducing or nonreducing conditions using Coomassie blueor, silver stain.

In certain embodiments, the antibody against Loricrin is a fragment thatspecifically binds Loricrin. An “antibody fragment” is a portion of anintact antibody that includes an antigen binding site of the intactantibody and thus retaining the ability to bind Loricrin. Antibodyfragments include:

-   -   (i) Fab fragments, having V_(L), C_(L), V_(H) and CH1 domains;    -   (ii) Fab′ fragments, which is a Fab fragment having one or more        cysteine residues at the C-terminus of the CH1 domain;    -   (iii) Fd fragments having V_(H) and CH1 domains;    -   (iv) Fd′ fragments having V_(H) and CH1 domains and one or more        cysteine residues at the C-terminus of the CH1 domain;    -   (v) Fv fragments having the V_(L) and V_(H) domains of a single        arm of an antibody;    -   (vi) dAb fragments (Ward et al., Nature 341, 544-546 (1989))        which consist of a VH domain;    -   (vii) isolated CDR regions, including any one or more of SEQ ID        Nos 1 to 6;    -   (viii) F(ab′)₂ fragments, a bivalent fragment including two Fab′        fragments linked by a disulphide bridge at the hinge region;    -   (ix) single chain antibody molecules (e.g. single chain Fv;        scFv) (Bird et al, Science 242:423-426 (1988); and Huston et        al., PNAS (USA) 85:5879-5883 (1988));    -   (x) “diabodies” with two antigen binding sites, comprising a        heavy chain variable domain (VH) connected to a light chain        variable domain (VL) in the same polypeptide chain (see, e.g.,        EP 404,097 and WO 93/11161;    -   (xi) “linear antibodies” comprising a pair of tandem Fd segments        (VH-CH1-VH-CH1) which, together with complementary light chain        polypeptides, form a pair of antigen binding regions (Zapata et        al. Protein Eng. 8 (10): 1057-1062 (1995); and U.S. Pat. No.        5,641,870).

Typically, the antibody against Loricrin is a recombinant monoclonalantibody. A “recombinant monoclonal antibody” is an antibody or antibodyfragment produced using recombinant antibody coding genes. In certainembodiments, the antibodies of the invention are generated from a humancombinatorial antibody library (e.g. HuCAL, BioRad).

Typically, the antibody against Loricrin is a monovalent Fab or bivalentFab fragment. A “bivalent Fab fragment” may be considered as equivalentto a F(ab′)2 fragment and formed via dimerization. For example, abivalent Fab fragment is formed via dimerization of a synthetic doublehelix loop helix motif (dHLX) or a bacterial alkaline phosphatase (AP)domain. In certain embodiments, the antibody against Loricrin comprisesa dimerization domain sequence and one or more linker sequences.

In certain embodiments, the antibody against Loricrin is a recombinantmonoclonal antibody fragment converted into an immunoglobulin (Ig)format. For example, when an Fc region is required, the variable heavyand light chain genes may be cloned into vectors with the desiredconstant regions and co-transfected for expression in mammalian cellsusing methods known to those skilled in the art. In certain embodiments,antibody fragments are converted to human IgA, IgE, IgG1, IgG2, IgG3,IgG3 or IgM.

In certain embodiments, the antibody against Loricrin is labelled withat least one epitope tag. Typical epitope tags include His6, Flag (e.g.DYKDDDDK), V5 (e.g. GKPIPNPLLGLDST), Strep (e.g. WSHPQFEK) and/or anycombination thereof. Typically, the antibody against Loricrin is amonovalent Fab or bivalent Fab fragment with one or more (e.g. two)epitopes.

In certain embodiments, the antibody against Loricrin is conjugated toan enzyme and/or fluorescent label.

In certain embodiments, the antibody specifically binds to Loricrin. Inother words, the antibodies against Loricrin bind Loricrin with abinding dissociation equilibrium constant (K_(D)) of less than about 30nM, less than about 20 nM, less than about 10 nm, less than about 1 nmor less than about 200 μm. The skilled person would understandtechniques for measuring binding strengths (e.g. koff-ratedetermination; ‘secondary screening’) include, for example, Bio-LayerInterferometry (e.g. using the Pall ForteBio Octet® System).

In certain embodiments, the antibody against Loricrin comprises thefollowing heavy chain variable domain complementarity determiningregions (CDRs):

(a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 22;(b) HCDR2 comprising the amino acid sequence of SEQ ID NO: 23; and(c) HCDR3 comprising the amino acid sequence of SEQ ID NO: 24.

In certain embodiments, the antibody against Loricrin further comprisesthe following light chain variable domain CDRs:

(d) LCDR1 comprising the amino acid sequence of SEQ ID NO: 25(e) LCDR2 comprising the amino acid sequence of SEQ ID NO: 26; and(f) LCDR3 comprising the amino acid sequence of SEQ ID NO: 27.

As used herein, the term “Complementarity Determining Regions” (CDRs)refers to the amino acid residues of an antibody variable domain thepresence of which are necessary for antigen binding. Each variabledomain typically has three CDR regions identified as CDR1, CDR2 andCDR3. Each complementarity determining region may comprise amino acidresidues from a “complementarity determining region” as defined by Kabat(i.e. about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the lightchain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in theheavy chain variable domain; Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)) and/or those residues from a“hypervariable loop” (i.e. about residues 26-32 (L1), 50-52 (L2) and91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2)and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). In some instances, a CDR region caninclude amino acids from both a CDR region defined according to Kabatand a hypervariable loop.

Unless otherwise specified herein, numbering of amino acid residues inthe Fc region or constant region is according to the EU numberingsystem, also called the EU index, as described in Kabat et al.

In certain embodiments, the antibody against Loricrin further comprisesthe following heavy chain variable domain framework regions (FRs):

(a) HCFR1 comprising the amino acid sequence of SEQ ID NO:28;(b) HCFR2 comprising the amino acid sequence of SEQ ID NO:29;(c) HCFR3 comprising the amino acid sequence of SEQ ID NO: 30; and(d) HCFR4 comprising the amino acid sequence of SEQ ID NO: 31.

In certain embodiments, the antibody against Loricrin further comprisesthe following light chain variable domain FRs:

(e) LCFR1 comprising the amino acid sequence of SEQ ID NO:32;(f) LCFR2 comprising the amino acid sequence of SEQ ID NO:33;(g) LCFR3 comprising the amino acid sequence of SEQ ID NO: 34; and(h) LCFR4 comprising the amino acid sequence of SEQ ID NO: 35.

As used herein, “Framework regions (FRs)” are those variable domainresidues other than the CDR residues. Each variable domain typically hasfour FRs identified as FR1, FR2, FR3 and FR4. If the CDRs are definedaccording to Kabat, the light chain FR residues are positioned at aboutresidues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4)and the heavy chain FR residues are positioned about at residues 1-30(HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) in the heavychain residues.

If the CDRs comprise amino acid residues from hypervariable loops, thelight chain FR residues are positioned about at residues 1-25 (LCFR1),33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the light chain andthe heavy chain FR residues are positioned about at residues 1-25(HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in the heavychain residues. In some instances, when the CDR comprises amino acidsfrom both a CDR as defined by Kabat and those of a hypervariable loop,the FR residues will be adjusted accordingly. For example, when CDRH1includes amino acids H26-H35, the heavy chain FR1 residues are atpositions 1-25 and the FR2 residues are at positions 36-49.

In certain embodiments, the antibody against Loricrin further comprisesan antibody variable domain comprising:

(a) a V_(H) sequence having at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or more sequence identity to theamino acid sequence of SEQ ID NO: 36;(b) a V_(L) sequence having at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or more sequence identity to theamino acid sequence of SEQ ID NO 37; or(c) a V_(H) sequence as in (a) and a V_(L) sequence as in (b).

In certain embodiments, the antibody against Loricrin comprises:

(d) a V_(H) sequence comprising SEQ ID NO:36;(e) a V_(L) sequence comprising SEQ ID NO: 37; or(f) a V_(H) sequence as in (d) and a V_(L) sequence as in (e).

As used herein, “antibody variable domain” refers to the portions of thelight and heavy chains of antibody molecules that include amino acidsequences of the CDRs (i.e., CDR1, CDR2, and CDR3) and the FRs (i.e.,FR1, FR2, FR3 and FR4). V_(H) refers to the variable domain of the heavychain. V_(L) refers to the variable domain of the light chain.

As used herein, “sequence identity” refers to a sequence having thespecified percentage of amino acid residues that are the same, whencompared and aligned (introducing gaps, if necessary) for maximumcorrespondence, not considering any conservative amino acidsubstitutions as part of the sequence identity. The percent identity canbe measured using sequence comparison software or algorithms or byvisual inspection. Various algorithms and software are known in the artthat can be used to obtain alignments of amino acid sequences. Suitableprograms to determine percent sequence identity include for example theBLAST suite of programs available from the U.S. Government's NationalCenter for Biotechnology Information BLAST web site. Comparisons betweentwo sequences can be carried using either the BLASTN or BLASTPalgorithm. BLASTN is used to compare nucleic acid sequences, whileBLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2(Genentech, South San Francisco, Calif.) or MegAlign, available fromDNASTAR, are additional publicly available software programs that can beused to align sequences. One skilled in the art can determineappropriate parameters for maximal alignment by alignment software. Incertain embodiments, the default parameters of the alignment softwareare used.

In certain embodiments, the antibody against Loricrin comprises a Fabfragment comprising the Fd chain of SEQ ID NO: 38; and/or the lightchain of SEQ ID NO: 39. Typically, such antibodies further comprise oneor more dimerization domain sequences, one or more linker sequencesand/or one or more epitope tags as described herein.

The invention provides use of antibodies against Loricrin as describedherein. Typically, the antibodies against Loricrin are used in themethods described herein of determining whether a subject with melanomahas an increased risk of metastasis.

The invention also provides antibodies that compete for binding toLoricrin with antibodies as described herein. Typically, competitionassays are used to identify an antibody that competes for binding toLoricrin. In an exemplary competition assay, immobilized Loricrin isincubated in a solution comprising a first labelled antibody that bindsto Loricrin and a second unlabelled antibody that is being tested forits ability to compete with the first antibody for binding to Loricrin.The second antibody may be present in a hybridoma supernatant. As acontrol, immobilized Loricrin may be incubated in a solution comprisingthe first labelled antibody but not the second unlabelled antibody.After incubation under conditions permissive for binding of the firstantibody to Loricrin excess unbound antibody may be removed, and theamount of label associated with immobilized Loricrin measured. If theamount of label associated with immobilized Loricrin is substantiallyreduced in the test sample relative to the control sample, then thatindicates that the second antibody is competing with the first antibodyfor binding to Loricrin. See, e.g., Harlow et al. Antibodies: ALaboratory Manual. Ch. 14 (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1988).

In certain embodiments, antibodies that compete for binding to Loricrinbind to the same epitope (e.g., a linear or a conformational epitope) asthe antibodies described herein. Detailed exemplary methods for mappingan epitope to which an antibody binds are provided in Morris “EpitopeMapping Protocols,” in Methods in Molecular Biology Vol. 66 (HumanaPress, Totowa, N.J., 1996).

Certain aspects of the present invention further provide isolatednucleic acids that encode any of the antibodies described herein. Alsoprovided is a vector (e.g., an expression vector) comprising the nucleicacid for expressing any of the antibodies described herein. Alsoprovided are host cells comprising the preceding nucleic acids and/orvectors.

Certain aspects of the present invention further provideimmunoconjugates comprising any of the antibodies described hereinconjugated to one or more capture agents. As described herein, a captureagent typically comprises a binding and/or detection moiety (e.g. anenzyme and/or fluorescent label).

Certain aspects of the present invention further provide antibodies thatbind to the same epitope as the anti-Loricrin antibody as definedherein.

As used herein, the term “epitope” means a protein determinant capableof specific binding to an antibody. Typically, an epitope compriseschemically active surface groupings of molecules such as amino acids orsugar side chains usually having specific three-dimensional structuraland charge characteristics. The epitope may comprise amino acid residuesdirectly involved in the binding and optionally additional amino acidresidues that are not directly involved in the binding.

As used herein, an antibody that “specifically” binds to an epitoperefers to an antibody that recognizes the epitope while only havinglittle or no detectable reactivity with other portions of Ambra-1. Suchrelative specificity can be determined e.g. by competition assays,foot-printing techniques or mass spectrometry techniques as known in theart.

In certain embodiments, the epitope comprises peptide antigenicdeterminants within single peptide chains of Loricrin. In certainembodiments, the epitope comprises conformational antigenic determinantscomprising one or more contiguous amino acids on a particular chainand/or on spatially contiguous but separate peptide chains. In certainembodiments, the epitope comprises post-translational antigenicdeterminants comprising molecular structures (e.g. carbohydrate groups)covalently attached to Loricrin.

The epitope may comprise any suitable number and/or type of amino acids,in any suitable position as defined herein. For example, the epitope maycomprise about 10 to about 20 amino acids, typically about 15 aminoacids, in or more contiguous or non-contiguous locations with respect tothe amino acid sequence of Loricrin as set forth in SEQ ID NO: 42.

In certain embodiments, the invention provides antibodies that bind toLoricrin, wherein said antibodies specifically bind to a regioncomprising 8, 9, 10, 11, 12, 13, 14, 15 or more of the amino acids 2 to20 of human Loricrin sequence shown in SEQ ID NO: 42. Typically, suchantibodies specifically bind to a region comprising SYQKKQPTPGPPVDCVKTS(SEQ ID NO: 54).

In certain embodiments, the invention provides antibodies that bind toLoricrin, wherein said antibodies specifically bind to a regioncomprising 8, 9, 10, 11, 12, 13, 14, 15 or more of the amino acids 73 to91 of human Loricrin sequence shown in SEQ ID NO: 42. Typically, suchantibodies specifically bind to a region comprising GGGGIGGPGGGSGGSVKYS(SEQ ID NO: 55). Typically, such antibodies specifically bind to a coreregion comprising at least VKYS (SEQ ID NO: 60).

In certain embodiments, the invention provides antibodies that bind toLoricrin, wherein said antibodies specifically bind to a regioncomprising 8, 9, 10, 11, 12 or more of the amino acids 95 to 109, 120 to134, 135 to 148, and/or 168 to 182 of human Loricrin sequence shown inSEQ ID NO: 42. Typically, such antibodies specifically bind to one ormore regions comprising GSSGGGSGCFSSGGG (SEQ ID NO: 56). Typically, suchantibodies specifically bind to one or more core regions comprising atleast CFS (SEQ ID NO: 61).

In certain embodiments, the invention provides antibodies that bind toLoricrin, wherein said antibodies specifically bind to a regioncomprising 8, 9, 10, 11, 12, 13, 14, 15 or more of the amino acids 262to 280 of human Loricrin sequence shown in SEQ ID NO: 42. Typically,such antibodies specifically bind to a region comprisingGIGSGCIISGGGSVCGGGS (SEQ ID NO: 57).

In certain embodiments, the invention provides antibodies that bind toLoricrin, wherein said antibodies specifically bind to a regioncomprising 8, 9, 10, 11, 12, 13, 14, 15 or more of the amino acids 279to 297 of human Loricrin sequence shown in SEQ ID NO: 42. Typically,such antibodies specifically bind to a region comprisingGSSGGGGGGSSVGGSGSGK (SEQ ID NO: 58).

In certain embodiments, the invention provides antibodies that bind toLoricrin, wherein said antibodies specifically bind to a regioncomprising 5, 6, 7, 8 or more of the amino acids 298 to 308 of humanLoricrin sequence shown in SEQ ID NO: 42. Typically, such antibodiesspecifically bind to a region comprising GVCICHQTQQK (SEQ ID NO: 59).

In certain embodiments, the antibody specifically binds to:

-   -   (i) amino acids 2-20 of human Loricrin (SEQ ID NO:42);    -   (ii) amino acids 73-91 of human Loricrin (SEQ ID NO:42);    -   (iii) amino acids 95-109 of human Loricrin (SEQ ID NO:42);    -   (iv) amino acids 120-134 of human Loricrin (SEQ ID NO:42);    -   (v) amino acids 135-148 of human Loricrin (SEQ ID NO:42);    -   (vi) amino acids 168-182 of human Loricrin (SEQ ID NO:42);    -   (vii) amino acids 262-280 of human Loricrin (SEQ ID NO: 42);    -   (viii) amino acids 279-297 of human Loricrin (SEQ ID NO: 42);        and/or    -   (ix) amino acids 298-308 of human Loricrin (SEQ ID NO: 42).

In certain embodiments, the invention provides a method of labellingLoricrin in a tissue sample overlying a primary melanoma, the methodcomprising:

-   -   (a) contacting the tissue sample with a monoclonal antibody        against Loricrin as defined herein; and    -   (b) visualising the antibody against Loricrin in the tissue        sample with a reagent that generates a detectable signal.

The tissue sample may comprise at least a portion of a peri-tumoralepidermis overlying the primary melanoma. Typically, the methodcomprises determining the expression of Loricrin in the stratum corneumof the epidermis. Typically, the tissue sample is a biopsy, or a sectionthereof, obtained from a subject suffering from melanoma.

Antibodies Against Ambra-1

In certain embodiments, the methods for determining whether a subjectwith melanoma has an increased risk of metastasis further comprisedetermining the expression level of Ambra-1 in the tissue sample.Typically, the expression level of Ambra-1 is determined using anantibody, e.g. a monoclonal antibody.

Antibodies against Ambra-1 include any antibodies as described above,including e.g. recombinant monoclonal antibodies.

In certain embodiments, the antibody against Ambra-1 is isolated.

In certain embodiments, the antibody against Ambra-1 is a fragment thatspecifically binds Ambra-1.

In certain embodiments, the antibody against Ambra-1 is labelled with atleast one epitope tag as described above. Typically, the antibodyagainst Ambra-1 is a monovalent Fab or bivalent Fab fragment with one ormore (e.g. two) epitopes as described above.

In certain embodiments, the antibody against Ambra-1 is conjugated to anenzyme and/or fluorescent label as described above.

In certain embodiments, the antibody specifically binds to Ambra-1. Inother words, the antibodies against Ambra-1 bind Ambra-1 with a bindingdissociation equilibrium constant (K_(D)) of less than about 30 nM, lessthan about 20 nM, less than about 10 nm, less than about 1 nm or lessthan about 200 pm. The skilled person would understand techniques formeasuring binding strengths include, for example, Bio-LayerInterferometry (e.g. using the Pall ForteBio Octet® System).

In certain embodiments, the method for determining whether a subjectwith melanoma has an increased risk of metastasis further comprises:

(iii) determining the expression level of Ambra-1 in the tissue sampleusing a monoclonal antibody against Ambra-1; and(iv) comparing the expression level obtained in (iii) with a referencetissue or levels obtained therefrom,wherein a decrease in the expression of Loricrin and Ambra-1 in thetissue sample compared to the reference tissue or levels, or a loss ofexpression of Loricrin and Ambra-1 in the tissue, is indicative of anincreased risk of metastasis.

In certain embodiments, the method of determining the expression ofAmbra-1 in the tissue sample comprises:

-   -   (a) contacting the tissue sample with the antibody against        Ambra-1; and    -   (b) visualising the antibody in the tissue sample with a reagent        that generates a detectable signal.

Typically, the tissue sample comprises keratinocytes overlying theprimary melanoma and the method comprises determining the expression ofAmbra-1 in the keratinocytes.

In certain embodiments, the antibody against Ambra-1 comprises thefollowing heavy chain variable domain complementarity determiningregions (CDRs):

(a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 1;(b) HCDR2 comprising the amino acid sequence of SEQ ID NO: 2; and(c) HCDR3 comprising the amino acid sequence of SEQ ID NO: 3.

In certain embodiments, the antibody against Ambra-1 further comprisesthe following light chain variable domain CDRs:

(d) LCDR1 comprising the amino acid sequence of SEQ ID NO: 4;(e) LCDR2 comprising the amino acid sequence of SEQ ID NO: 5; and(f) LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.

In certain embodiments, the antibody against Ambra-1 further comprisesthe following heavy chain variable domain framework regions (FRs):

(a) HCFR1 comprising the amino acid sequence of SEQ ID NO: 7;(b) HCFR2 comprising the amino acid sequence of SEQ ID NO: 8;(c) HCFR3 comprising the amino acid sequence of SEQ ID NO: 9; and(d) HCFR4 comprising the amino acid sequence of SEQ ID NO: 10.

In certain embodiments, the antibody against Ambra-1 further comprisesthe following light chain variable domain FRs:

(e) LCFR1 comprising the amino acid sequence of SEQ ID NO: 11;(f) LCFR2 comprising the amino acid sequence of SEQ ID NO: 12;(g) LCFR3 comprising the amino acid sequence of SEQ ID NO: 13; and(h) LCFR4 comprising the amino acid sequence of SEQ ID NO: 14.

In certain embodiments, the antibody against Ambra-1 further comprisesan antibody variable domain comprising:

(a) a V_(H) sequence having at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or more sequence identity to theamino acid sequence of SEQ ID NO: 15;(b) a V_(L) sequence having at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or more sequence identity to theamino acid sequence of SEQ ID NO 16; or(c) a V_(H) sequence as in (a) and a V_(L) sequence as in (b).

In certain embodiments, the antibody against Ambra-1 comprises:

(d) a V_(H) sequence comprising SEQ ID NO:15;(e) a V_(L) sequence comprising SEQ ID NO: 16; or(f) a V_(H) sequence as in (d) and a V_(L) sequence as in (e).

In certain embodiments, the antibody against Ambra-1 comprises a Fabfragment comprising:

-   -   (a) the Fd chain of SEQ ID NO: 17;    -   (b) the light chain of SEQ ID NO: 18;    -   (c) a dimerization domain sequence;    -   (d) one or more linker sequences; and/or    -   (e) one or more epitope tags.

The invention provides use of antibodies against Ambra-1 as describedherein in methods of determining whether a subject with melanoma has anincreased risk of metastasis. The antibodies against Ambra-1 may be usedin any method, assay or kit described herein.

The invention also provides antibodies that compete for binding toAmbra-1 with antibodies as described herein.

The invention further provides antibodies that bind to the same epitopeas the anti-Ambra-1 antibody as defined herein. In certain embodiments,the invention provides antibodies that bind specifically to peptidesnear the carboxyl-terminus of human Ambra-1.

In certain embodiments, the epitope comprises peptide antigenicdeterminants within single peptide chains of Ambra-1. In certainembodiments, the epitope comprises conformational antigenic determinantscomprising one or more contiguous amino acids on a particular chainand/or on spatially contiguous but separate peptide chains. In certainembodiments, the epitope comprise post-translational antigenicdeterminants comprising molecular structures (e.g. carbohydrate groups)covalently attached to Ambra-1.

The epitope may comprise any suitable number and/or type of amino acids,in any suitable position as defined herein. For example, the epitope maycomprise about 3 to about 10 amino acids, typically about 3 to about 8amino acids, in or more contiguous or non-contiguous locations withrespect to the amino acid sequence of Ambra-1 as set forth in SEQ ID NO:21, 46, or 48.

In certain embodiments, the invention provides antibodies that bind toAmbra-1, wherein said antibodies specifically bind to a regioncomprising 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more ofthe amino acids 1270 to 1298 of human Ambra-1 sequence shown in SEQ IDNO: 21. Typically, such antibodies specifically bind to a regioncomprising amino acids 1280 to 1281 of SEQ ID NO: 21, amino acids 1294to 1296 of SEQ ID NO: 21 and/or amino acids 1294 to 1297 of SEQ ID NO:21.

In certain embodiments, the invention provides antibodies that bind toAmbra-1, wherein said antibodies specifically bind to a regioncomprising 8, 9, 10, 11, 12, 13, 14, 15 or more of CGGSSRGDAAGPRGEPRNR(SEQ ID NO: 46). Typically, such antibodies specifically bind to aregion comprising at least EPRN (SEQ ID NO: 52) or at least EPR (SEQ IDNO: 53) of Ambra-1.

In certain embodiments, the invention provides antibodies that bind toAmbra-1, wherein said antibodies specifically bind to a regioncomprising 8, 9, 10, 11, 12, 13, 14, 15 or more of DGGSSRGDAAGPRGEPRNR(SEQ ID NO: 49). Typically, such antibodies specifically bind to aregion comprising DG, EPRN (SEQ ID NO: 52) and/or EPR (SEQ ID NO: 53) ofAmbra-1.

In certain embodiments, the invention provides antibodies that bind toAmbra-1, wherein said antibodies specifically bind to a regioncomprising HLLDGGSSR (SEQ ID NO: 50) and/or EPR (SEQ ID NO: 53) ofAmbra-1.

In certain embodiments, the invention provides antibodies that bind toAmbra-1, wherein said antibodies specifically bind to a regioncomprising NHLLDGGSSR (SEQ ID NO: 51) and/or EPR (SEQ ID NO: 53) ofAmbra-1.

In certain embodiments, the invention provides antibodies that bind to apeptide fragment consisting of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 or more of the amino acids 1270 to 1298 of human Ambra-1 sequenceshown in SEQ ID NO: 21. Typically, the invention provides antibodiesthat bind specifically to SEQ ID NO: 46.

In certain embodiments, the invention provides antibodies thatspecifically bind to Ambra-1, wherein said antibody specifically bindsto:

-   -   (i) amino acids 1280-1281 and/or 1294-1296 of human Ambra-1 (SEQ        ID NO:21);    -   (ii) amino acids 1277-1285 and/or 1294-1296 of human Ambra-1        (SEQ ID NO:21);    -   (iii) amino acids 1276-1285 and/or 1294-1296 of human Ambra-1        (SEQ ID NO:21);    -   (iii) amino acids 1280-1296 of human Ambra-1 (SEQ ID NO:21);    -   (iv) amino acids 1276-1296 of human Ambra-1 (SEQ ID NO: 21)    -   (v) amino acids 1280-1298 of human Ambra-1 (SEQ ID NO:21);        and/or    -   (vi) amino acids 1276-1298 of human Ambra-1 (SEQ ID NO:21.

In certain embodiments, the invention provides a method of labellingAmbra-1 in a tissue sample overlying a primary melanoma, the methodcomprising:

-   -   (a) contacting the tissue sample with a monoclonal antibody        against Ambra-1 as defined herein; and    -   (b) visualising the antibody against Ambra-1 in the tissue        sample with a reagent that generates a detectable signal.

The tissue sample may comprise keratinocytes overlying the primarymelanoma and the method comprises determining the expression of Ambra-1in the keratinocytes.

Unexpectedly, the combination of consistent and distinct expressionpatterns of the anti-Loricrin and Ambra-1 antibodies describes hereinallows rapid and accurate identification of subjects having decreased orloss of expression of Loricrin and/or Ambra-1. Thus, the combined use ofthe anti-Loricrin and Ambra-1 antibodies provides improved methods fordetermining whether a subject with melanoma has an increased risk ofmetastasis.

Methods of Treatment

Certain aspects of the present invention provide methods of determininga treatment regime for a subject suffering from melanoma, comprisingdetermining the expression of Loricrin (and optionally Ambra-1) asdescribed herein.

Aptly, the method further comprises comparing the expression of Loricrin(and optionally Ambra-1) as described herein.

If expression of Loricrin (and optionally Ambra-1) is normal orincreased, a normal recognized care pathway may be followed.

A “normal recognized care pathway”, as will be known to those skilled inthe art, will be understood as meaning that a wider excision of the scarleft by excision of the primary melanoma is carried out on the subject.The size of the wider excision will be determined by a clinician orsurgeon, based on the Breslow depth of the primary melanoma. A normalrecognized care pathway may further comprise regular (e.g. every 3-12months) clinical assessment of the subject for up to 5 years. Inembodiments where the primary melanoma is stage 2b or 2c, the normalrecognized care pathway may further comprise carrying out a staging CTscan on the subject, from the head to the pelvis, at the time ofdiagnosis. Some treatment centers offer staging sentinel lymph nodebiopsy of all stage 2a, 2b and 2c tumors. Thus, in some embodiments, thenormal recognized care pathway may further comprise carrying out asentinel lymph node biopsy.

If expression of Loricrin (and optionally Ambra-1) is decreased or lost,a systemic anti-cancer treatment regime may be followed.

In some embodiments, a systemic anti-cancer treatment regime comprisesadministering a therapeutic agent to the subject. Typically, thetherapeutic agent is capable of inhibiting, preventing or delayingmetastasis of melanoma in the subject.

In some embodiments, the therapeutic agent is a chemotherapeutic agent.Any suitable chemotherapeutic agent may be administered to the subject.As used herein, a “chemotherapeutic agent” means any therapeutic agentuseful for the treatment of cancer, and encompasses small molecules aswell as biological agents, such as antibodies.

In some embodiments, the chemotherapeutic agent is selected fromDacarbazine (DTIC), Temozolomide, Nab-paclitaxel, Paclitaxel, Carmustine(BCNU), Cisplatin, Carboplatin, Vinblastine, interleukin 2, interferonalpha, or any combination thereof.

In some embodiments, the chemotherapeutic agent is a biological agent,such as an anti-PD and/or anti-CTLA4 therapy. For example, thebiological agent may be selected from ipilumab, nivolumab, pembrolizumaband/or any combination thereof. For example, the biological agent may beselected from nivolumab and ipilimumab. In some embodiments, thebiological agent is a B-Raf inhibitor, such as vemurafenib and/ordabrafenib. In certain embodiments, the biological agent is nivolumaband/or ipilimumab; dabrafenib and/or trametinib; vemurafenib and/orcobimetinib; and/or any combination thereof.

Thus, in certain embodiments the invention provides a method of treatinga subject suffering from melanoma comprising administering a therapeuticagent to a subject identified as having decreased or loss of expressionof Loricrin (and optionally Ambra-1) by methods described herein.

In certain embodiments, the invention provides a therapeutic agent asdescribed herein for use in a method of treating melanoma in a subject,wherein said subject has been identified as having an increased risk ofmetastasis according to any method described herein.

Ideally, a subject identified as having an increased risk of metastasisis treated as soon as possible to minimize the chances of development ofmetastasis. Thus, in some embodiments the method or treatment regime isfor preventing, inhibiting or delaying metastasis or decreasing the riskof metastasis in the subject.

In some embodiments, a subject is treated immediately or shortly afterbeing identified as having an increased risk of metastasis.

In some embodiments, treatment with the therapeutic agent is carried outafter surgery to excise the primary melanoma.

In some embodiments, a method of treatment or a treatment regime mayfurther include one or more of: intensified imaging (e.g. CT scan, PET,MRI, X-ray) of the subject; discussion and/or offering of, or carryingout, a sentinel lymph node biopsy; partial or complete lymphadenectomy;inclusion of the subject in clinical trials; and radiation therapy.

In some embodiments, a therapeutic agent is administered to the subjectno more than 12 weeks, no more than 10 weeks, no more than 6 weeks, nomore than 4 weeks, no more than 2 weeks or no more than 1 week after thesubject is identified as having a decrease or loss of expression ofLoricrin in the tissue sample.

Non-limiting routes of administration of the therapeutic agent includeby oral, intravenous, intraperitoneal, subcutaneous, intramuscular,topical, intradermal, intranasal or intrabronchial administration (forexample as effected by inhalation). In some embodiments, the therapeuticagent is administered parenterally, e.g., intravenously. Common modes ofadministration by which the therapeutic agent may be administeredinclude, for example, administration as a bolus dose or as an infusionover a set period.

A therapeutic agent may be administered in an amount effective toprevent, inhibit or delay the development of metastasis.

Suitable doses and dosage regimes for a given subject and therapeuticagent can be determined using a variety of different methods, such asbody-surface area or body-weight, or in accordance with specialistliterature and/or individual hospital protocols. Doses may be furtheradjusted following consideration of a subject's neutrophil count, renaland hepatic function, and history of any previous adverse effects to thetherapeutic agent. Doses may also differ depending on whether atherapeutic agent is used alone or in combination.

The skilled person will recognize that further modes of administration,dosages of therapeutic agents and treatment regimens can be determinedby the treating physician according to methods known in the art.

Assays and Kits

Certain aspects of the present invention provide in vitro assays forpredicting an increased risk of metastasis in a subject suffering frommelanoma.

Aptly, the assay comprises contacting a tissue sample overlying aprimary melanoma with an antibody against Loricrin as described herein.In the assay, the presence of Loricrin creates a Loricrin-antibodycomplex. The assay may further comprise detecting and/or quantifying theLoricrin-antibody complex.

Typically, the step of detecting and/or quantifying theLoricrin-antibody complex comprises contacting the tissue sample(s) (orthe section(s) or portion(s) thereof) with at least one capture agent.Typically, a capture agent which binds specifically to the antibody ofthe invention is used to detect and/or quantify the Loricrin-antibodycomplex.

In some embodiments, the capture agent comprises a binding moiety and adetection moiety.

In some embodiments, the binding moiety is a secondary antibody whichbinds specifically to the antibody against Loricrin. For example, thebinding moiety may be a universal anti-IgG antibody that is capable ofbinding to the antibody against Loricrin (e.g. a flag sequence of theantibody).

In some embodiments, the binding moiety is an enzyme (e.g. alkalinephosphatase) which binds specifically to the antibody against Loricrin.

In some embodiments, the method further comprises one or more wash stepsto remove unbound antibodies and, optionally, unbound capture agents.

Typically, a suitable detection moiety is selected from a fluorescentmoiety, a luminescent moiety, a bioluminescent moiety, a radioactivematerial, a colorimetric moiety, a nanoparticle having suitabledetectable properties, a chromogenic moiety, biotin or an enzyme.

Suitable fluorescent moieties include fluorescent proteins (such asphycoerythrin (PE), peridinin-chlorophyll-protein complex (PerCP) andallophycocyanin (APC)) fluorescent dyes (such as FluoresceinIsothiocyanate (FITC), rhodamines (Rs) and cyanines (Cys)), fluorescenttandem complexes (such as Allophycocyanin-Cyanine 7 (APC-Cy7),Peridinin-Chlorophyll-Protein complex-Cyanine 5 (PerCP-cy5) andPhycoerythrin-Texas Red (PE-TexasRed)), and nanocrystals (such as QDot525, QDot 545 and QDot 625). The presence of Loricrin-antibody complexescan be detected using fluorescence microscopy via the use of fluorescentligands or a capture agent comprising a fluorescent detection moiety.

In embodiments where the detection moiety comprises an enzyme, thepresence of the Loricrin-antibody complex can be detected and/orquantified by detecting and/or quantifying the reaction product of areaction of a substrate catalyzed by the enzyme. In these embodiments,the method further comprises adding a substrate of the enzyme anddetecting and/or quantifying the product of the reaction performed onthe substrate by the enzyme. For example, the reaction may result in theproduction of a colored precipitate, which would be detected using lightmicroscopy.

Suitable enzymes include, for example, alkaline phosphatase andhorseradish peroxidase. A chromogenic substrate of alkaline phosphataseis PNPP (p-Nitrophenyl Phosphate, Disodium Salt). PNPP produces a yellowwater-soluble reaction product that absorbs light at 405 nm. Chromogenicsubstrates of horseradish peroxidase include ABTS (2,2′-Azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt), which yieldsa green reaction product, OPD (o-phenylenediamine dihydrochloride) whichyields a yellow-orange reaction product, and TMB(3,3′,5,5′-tetramethylbenzidine) soluble substrates yield a blue colourwhen detecting HRP. Other suitable enzyme-substrate combinations,methods of detecting the Loricrin-antibody complexes, and suitabledetection moieties will be known to those skilled in the art.

In certain embodiments, the antibody against Loricrin or the captureagent is immobilized on a solid phase surface, for example a microarray,slide, well or bead.

In certain embodiments, the expression of Loricrin is detected and/orquantified by visual assessment, for example, microscopy. In otherembodiments, the expression of Loricrin is detected and/or quantified byan automated slide scanner.

In certain embodiments, the method of detecting and/or quantifying theLoricrin-antibody complex comprises outputting, optionally on acomputer, an indication of whether the one or more complexes are presentor absent, and this indicates whether the subject suffering frommelanoma has an increased risk of metastasis.

In certain embodiments, the invention further comprises contacting thetissue sample with an antibody against Ambra-1 as described herein,where the presence of Ambra-1 creates an Ambra-1-antibody complex anddetecting and/or quantifying the Ambra-1-antibody complex. TheAmbra-1-antibody complex may be detected and/or quantified as describedherein for the Loricrin-antibody complex.

The invention also provides a kit for predicting an increased risk ofdeveloping metastasis of a subject suffering from melanoma, the kitcomprising an antibody against Loricrin as described herein. In someembodiment, the kit further comprises an antibody against Ambra-1 asdescribed herein.

In certain embodiments, the kit further comprises instructions for usingthe kit to predict the risk of metastasis in a subject suffering frommelanoma.

In certain embodiments, the kit further comprises at least one captureagent. Typically, a capture agent comprises a detection moiety and/or abinding moiety as described herein.

In certain embodiments, the detection moiety is an enzyme (e.g. alkalinephosphatase) and the kit further comprises a substrate of the enzyme.

Typically, the kit may further comprise one or more additionalcomponents such as reagents and/or apparatus necessary for carrying outan in vitro assay, e.g. buffers, fixatives, wash solutions, blockingreagents, diluents, chromogens, enzymes, substrates, test tubes, plates,pipettes etc.

The kit of certain embodiments of the invention may advantageously beused for carrying out a method of certain embodiments of the inventionand could be employed in a variety of applications, for example in thediagnostic field or as a research tool. It will be appreciated that theparts of the kit may be packaged individually in vials or in combinationin containers or multi-container units. Typically, manufacture of thekit follows standard procedures which are known to the person skilled inthe art.

EXAMPLES Example 1—Production of Anti-Ambra-1 and Anti-LoricrinAntibodies

Anti-Ambra-1 and Loricrin antibodies were produced using BioRad HuCALPLATINUM® antibody generation technology and CysDisplay® technology.HUCAL stands for ‘Human Combinatorial antibody library’, which is asynthetic (generated by de novo gene synthesis) antibody librarycontaining human antibody gene sequences covering more than 95% of thehuman structural gene repertoire (45 billion antibodies) that are clonedin E. coli phagemid vectors. Each E. coli phage contains one of the 45billion antibody genes and displays the corresponding antibody on theirsurface in Fab format, by means of a disulfide linkage between Fab andgene III protein (Cys Display).

Antigens of Ambra-1 (a synthesized peptide) and Loricrin (native humanantigen) were used to isolate the antibodies described herein. Theantigens were immobilized on to a solid support (i.e., ELISA microtiterplates or covalently coupled to magnetic beads), before the HuCALlibrary presented on phage was incubated with the antigens. Non-specificantibodies were removed by washing and specific antibody phages elutedby adding a reducing agent.

CysDisplay technology, where the Fab antibody fragment is linked to thephage by a disulphide bond that is easily cleavable rather than aconventional peptide bond, was used to allow more efficient elution ofhigh affinity phages with reducing agents during antibody selection(Bio-Rad). This ensured that high affinity antibodies were not lostduring selection, a common problem with more traditional panning phagedisplay methods.

The specific antibody phages were used to infect an E. coli culturealong with helper phages, allowing the enriched antibody phage libraryto be used for subsequent rounds of panning (usually 2-3 rounds ofenrichment panning).

After panning, the phagemid DNA encoding the enriched antibodypopulation was isolated as a pool and subcloned into a Fab expressionvector containing antibiotic resistance. The vector format chosen was abivalent Fab (Fab-A-FH) formed with dimerization of bacterial alkalinephosphatase, with two tags Flag (DYKDDDK) and His 6 (His6). E. coli wasthen transformed with the Fab expression vector ligation mixture andplated on agar plates containing antibiotic. Each growing colonyrepresents a monoclonal antibody and was picked and grown in a 384 wellmicrotiter plate. Antibody expression was induced and the cultureharvested and lysed to release the antibodies.

Culture lysates were screened primarily for specific antigen binding toantigens by indirect ELISA. 95 ELISA-positive antibody clones, derivedfrom the primary screening, were ranked according to their bindingstrength (koff-rate determination; ‘secondary screening’) as measured byBio-Layer Interferometry using the Pall ForteBio Octet® System.Antibodies were then selected according to both antigen specificity andbinding strength. Hits from the primary or secondary antibody screeningprocedures were sequenced to identify unique antibodies. The Fabantibodies with unique sequences were expressed in E. coli and purifiedusing one-step affinity chromatography. Purified antibodies were testedby QC ELISA for required specificity. This QC ELISA screen was performedon native as well as denatured antigen due to the final antibodyapplication of immunohistochemistry, where antigens during the tissueprocessing may be denatured, as well as immobilized control proteinsGlutathione S-transferase, BSA (carrier protein), N1-CD33-His6 (theectodomain of human CD33 fused to the N1 domain of the g3p filamentousphage M13) used for calculation of background. Purity was assessed byCoomassie® staining of a sodium dodecyl sulfate-polyacrylamide gel(SDS-PAGE) and concentration measured by UV absorbance at 280 nm.

13 Ambra-1 and 15 loricrin recombinant monoclonal antibody Fab fragmentswere obtained and used for subsequent validation (FIGS. 1 and 2).

Example 2—Validation of Anti-Ambra-1 and Anti-Loricrin Antibodies

The recombinant human HuCAL antibody fragment antibodies were validatedin the first instance using immunohistochemistry protocols on normalskin tissue (where Ambra-1 and Loricrin are expressed) and in aselection of AJCC stage I melanoma tumor tissue (where expression ofboth Ambra-1 and Loricrin is maintained in the epidermis overlying thetumor or lost). In all instances the staining of the HuCAL antibodieswere compared to commercially available Ambra-1 and Loricrin antibodiesby Abcam in the same tissue. Negative control of omitting the primaryantibody and using anti-Flag (HuCAL antibodies) or anti-Rabbit (Abcamantibodies) secondary antibodies was also included in each instance.

Materials and Methods

An optimised staining protocol for the Ambra-1 and Loricrin antibodiesis described briefly below:

-   -   Antigen retrieval buffer 10 mM Tris HCL Ph 9.    -   Positive control: normal skin section normal epidermis adjacent        to tumour in melanoma sections.    -   Negative controls: Null primaries of normal skin NH9808 and all        tumours.    -   Use research antibody for Loricrin Abcam Ab176322 1/500, 1 hr        room temperature, secondary anti-rabbit 1/200 from Rabbit        VECTASTAIN Elite ABC HRP Kit (VECTOR laboratories).    -   Use research antibody for Ambra-1 Abcam Ab69501 1/2500 1 hr room        temperature, secondary anti-rabbit 1/200 from Rabbit VECTASTAIN        Elite ABC HRP Kit (VECTOR laboratories).    -   Test conditions: Ambra-1 and Loricrin HUCAL antibodies chosen at        20 ug/ml 1 hr room temperature. Secondary antibody used Sigma        anti-flag HRP A8592 at 1/200 concentration as optimised for each        antibody.    -   All tissue sections formalin fixed paraffin embedded, cut at 5        μm and baked.

Deparaffinise Sections:

1. Place slides in metal rack and incubate in Histoclear 20 mins.2. Dip slides in: 100% ethanol for 5 secs, 75% for 5 secs, 50% for 5secs, dH2O for 5 secs.

Antigen Retrieval:

3. Place slides in plastic rack, with blank slides around the edges toeven out the heat.4. Place in buffer (10 Mm Tris HCl PH 9) and heat in microwave.5. Allow to cool slowly in buffer solution for 15 minutes.

Permeabilize Cells:

6. Place slides on flat metal tray, draw around sections withhydrophobic pen and allow to dry.7. Incubate sections with PBS/T (PBS+0.05% Tween 20) for 3 mins torehydrate sections.8. Incubate slides in 0.2% Triton X-100 in PBS/T for 10 mins.

Block Endogenous Peroxidase:

9. Wash with PBS/T10. Incubate with 3% H₂O₂ in water for 10 min

Endogenous Avidin/Biotin Blocking Step:

11. Wash with PBS/T12. Block endogenous Avidin (Vector Labs Avidin/Biotin Blocking kit) for15 mins13. Wash with PBS/T14. Block endogenous Biotin (Vector Labs Avidin/Biotin Blocking kit) for15 mins

Protein Blocking Step:

15. Wash with PBS/T16. Incubate with appropriate 2% serum in PBS/T (2 drops in 5 ml) Normalmouse/goat serum (Sigma/Vector Labs) for 20 mins17. Wash with PBS/T

Primary Antibody:

1. Incubate with anti-Loricrin/anti Ambra-1 primary antibody (in PBS/2%serum) 1 hr room temperature. Incubate positive control slide withresearch antibody abCam ab176322, ab69501 (PBS/2% serum) 1 hr roomtemperature.2. 3× Wash with PBS/T

Secondary Antibody:

3. Incubate with biotinylated anti rabbit/anti-flag secondary antibody.4. Prepare ABC Reagent from Vectastain Elite kit, leave at roomtemperature for 30 mina. 2.5 ml PBS+1 drop A+1 drop B5. 3× Wash with PBS/T

Staining:

6. Incubate with ABC reagent for 30 mins.7. 3× Wash with PBS/T.8. Prepare DAB substrate as per manufacturer's instructions (Vector,ImmPACT DAB EqV peroxidase substrate SK-4103). Mix an equal volume ofDAB reagent 1 and 2, mix well before use (working solution can be storedfor 1 week at 2-8° C.).9. Incubate tissue sections with DAB substrate for 2 mins.10. Rinse slides in water for 5 mins to stop peroxidase reaction.11. Incubate in Meyer's Haemalum (hematoxylin) for 10 minutes.12. Wash 10 min with frequent changes13. Put slides in: 75% ethanol for 5 secs (blot firmly), 100% ethanolfor 5 secs14. Put slides in Histoclear to clean sections for 2 minutes.15. Allow slides to dry and mount cover slips with DPX.

Results

Following immunohistochemical analysis, the recombinant human HuCALantibody fragment for Ambra-1 Abd33473.1 (33473) and Loricrin AbD33047.1(33047) were selected due to the physiologically relevant expressionpatterns and staining intensity.

Representative pictomicrographs are shown (see FIGS. 3 to 8),highlighting commercially available Abcam and the selected HuCALAbd33473.1 and AbD33047.1 antibody staining in normal skin and stage Imelanoma tumor tissue where expression of both Ambra-1 and Loricrinmaintained or lost in the epidermis overlying the tumor. Importantly,the selected HuCAL Abd33473.1 and AbD33047.1 antibodies led to a moreconsistent and distinct pattern of expression as compared to thecommercially available Abcam antibodies.

The HuCAL recombinant monoclonal Fab antibody against human Ambra-1 andLoricrin were converted into full length chimeric antibody with an IgG1mouse Fc region to allow detection of antibodies by standard automatedimmunohistochemistry. This was achieved by sub-cloning the heavy andlight chain genes of the Fab antibody into a vector with an IgG1 mouseconstant region, followed by transient expression in mammalian HKB-11cells, purification from HKB-11 supernatants by affinity chromatographyand subsequent quality control by ELISA assay (FIGS. 9 and 10).

The full-length chimeric immunoglobulin antibodies against Ambra-1 andLoricrin were validated by automated immunohistochemistry in a smallcohort of AJCC stage I melanoma tumors with Ambra-1 and Loricrinmaintained or lost in the epidermis overlying the tumor. Staining wasperformed using the Ventana Benchmark XT automated IHC staininginstrument (Ventana Medical Systems Inc.) with ultraView Universal DABDetection Kit (Ventana Medical Systems Inc.) or ultraView UniversalAlkaline Phosphatase Red Detection Kit, (Ventana Medical Systems Inc.)according to the manufacturer's specifications. In all instances, thestaining of the HuCAL antibodies were compared to commercially availableAmbra-1 and Loricrin antibodies by Abcam in the same tissue (FIGS. 11and 12).

In summary, the recombinant HuCAL antibody fragment Ambra-1 (AbD33473)stained keratinocytes cytoplasmically within the epidermis or normalskin/normal skin adjacent to the tumour, with intensity increasingupwards through the epidermis to the stratum corneum as expected in linewith keratinocyte differentiation. This expression was either maintainedor lost over the stage I melanoma tumour samples appropriately. Ambra-1AbD33473 also consistently stained tumor cells, in contrast to theresearch grade antibodies. Thus, the selected AbD33473 antibody iscapable of consistent and superior staining as compared to the researchgrade polyclonal antibodies.

Loricrin AbD33047 stained cytoplasmically in the uppermost layer (e.g.the most differentiated top layer) of keratinocytes in the stratumcorneum of the epidermis, presenting as a thin line in normalskin/normal skin adjacent to the tumor. This line was either maintainedor lost over the stage I melanoma tumor samples appropriately. Thus, theselected AbD33047 antibody allows improved detection of loricrin ascompared to the research grade polyclonal antibodies.

Example 3—Epitope Mapping of Anti-Ambra-1 Antibodies

Epitopes of Ambra-1 recognized by anti-Ambra-1 antibodies were mappedusing linear, conformational and replacement analysis epitope mapping(Pepscan Presto BV) using established techniques (Timmerman et al(2007). J. Mol. Recognit. 20: 283-299; Langedijk et al. (2011)Analytical Biochemistry 417: 149-155).

The anti-Ambra-1 antibody was tested on arrays with overlapping linearpeptides and looped peptides, based on the C-terminal sequence ofAmbra-1:

(SEQ ID NO: 27) VSLPSAEGPTLHCELTNNNHLLDGGSSRGDAAGPRGEPRNR

The core epitope, based on overlapping peptides in the linear and loopedarrays, was determined to be sequence ₃₇EPR₃₉. A second binding site isapparent only in specific length peptides, and more pronounced in thelooped peptide array. Binding to these residues may require a veryspecific conformation, which in the LOOP11 peptide mimics is providedreadily, and can only be induced with the specific residue content inthe LIN11 peptides. If so, this suggests that a secondary structure maybe formed for recognition of these residues. This recognition occursonly in this length peptide. Thus, it is possible that specific residuesneed to be aligned precisely to be recognized. This may represent astructure such as a beta turn, in which some residues on oppositestrands are required for the formation, allowing the proper positioningof the two identified residues in the loop tip. The location of the tworesidues in the center of the 11-mers also corroborates such apossibility. In conclusion, the main residues for binding are ₃₇EPR₃₉,which may be aided by ₂₃DG₂₄ in a specific conformation.

Details of the epitope information is summarized in the Table below.Core binding sites are listed based on overlap of peptides. Underlinedsequence highlights the key residues deducted from the replacementanalysis (REPNET):

Anti-Ambra-1 Epitope REPNET ₂₃ DGGSSRGDAAGPRGEPRNR₄₁ (SEQ ID NO: 28) LIN₃₇EPR₃₉ (SEQ ID NO: 31) ₁₉HLLDGGSSR₂₈ (SEQ ID NO: 29) LOOP₃₇EPR₃₉ (SEQ ID NO: 31) ₁₉NHLLDGGSSR₂₈ (SEQ ID NO: 30)

Example 4—Epitope Mapping of Anti-Loricrin Antibodies

Epitopes of Loricrin recognized by anti-Loricrin antibodies were mappedusing linear, conformational and replacement analysis epitope mapping(Pepscan Presto BV) using established techniques (Timmerman et al(2007). J. Mol. Recognit. 20: 283-299; Langedijk et al. (2011)Analytical Biochemistry 417: 149-155).

The anti-Loricrin antibody was tested on arrays with overlapping linearpeptides and looped peptides, based on the full length sequence ofLoricrin:

(SEQ ID NO: 42) MSYQKKQPTPQPPVDCVKTSGGGGGGGGSGGGGCGFFGGGGSGGGSSGSGCGYSGGGGYSGGGCGGGSSGGGGGGGIGGCGGGSGGSVKYSGGGGSSGGGSGCFSSGGGGSGCFSSGGGGSSGGGSGCFSSGGGGSSGGGSGCFSSGGGGFSGQAVQCQSYGGVSSGGSSGGGSGCFSSGGGGGSVCGYSGGGSGGGSGCGGGSSGGSGSGYVSSQQVTQTSCAPQPSYGGGSSGGGGSGGSGCFSSGGGGGSSGCGGGSSGIGSGCIISGGGSVCGGGSSGGGGGGSSVGGSG SGKGVPICHQTQQKQAPTWPSK

These studies identified a number of epitopes, as shown in the Tablebelow. Especially significant epitope candidates, for which the highestbinding signals were observed, are highlighted. Under the assumptionthat the binding intensity to the peptide models correlates with theimportance of the epitope candidate in a full-length protein context,this indicates them being the main interaction sequences. In case of73-GGGGIGGPGGGSGGSVKYS-91, double alanine mutation additionallyindicated an importance of residues VKYS. Notably, the sequence ofLoricrin includes a large degree of sequence similarity and recurringmotifs. This results in peptide mimics with similar sequence fordifferent regions (Seq #: 95-109; 120-134; 135-148; 168-182). Thisshould especially be considered for recurring core sequences with lowerintensity. These could potentially be so called secondary epitopes thatexhibit binding purely due to sequence similarities. Minor peaks thatfulfill these characteristics are indicated in the Table below. Therecurring sequences that exhibit binding capabilities might also be ofsupportive nature to antibody binding in a full length Loricrinconstruct. A decrease in binding to peptides where residues CFS (Seq #:103-105; 128-130; 143-145; 176-178) are mutated to alanine was observed,especially in case of 15-mer linear mimics. Together with thesignificant peak intensity and consecutive peptide sequences, thisserves as an indication that at least one of the repeating sequences ispointing towards an epitope. Interestingly, the described bindingsequence is not or to a varying degree observed in the conformationalpeptide mimics. This possibly points towards an epitope that does notadapt a defined secondary structure.

SEQ ID NO: 42 Epitope   2-20 SYQKKQPTPGPPVDCVKTS (SEQ ID NO: 54) Primary 73-91 GGGGIGGPGGGSGGSVKYS (SEQ ID NO: 55) Primary  95-109GSSGGGSGCFSSGGG (SEQ ID NO: 56) Recurring 120-134GSSGGGSGCFSSGGG (SEQ ID NO: 56) Recurring 135-148GSSGGGSGCFSSGGG (SEQ ID NO: 56) Recurring 168-182GSSGGGSGCFSSGGG (SEQ ID NO: 56) Recurring 262-280GIGSGCIISGGGSVCGGGS (SEQ ID NO: 57) 279-297GSSGGGGGGSSVGGSGSGK (SEQ ID NO: 58) Primary 298-308GVCICHQTQQK (SEQ ID NO: 59) Primary

The reader's attention is directed to all papers and documents which arefiled concurrently with or before this specification in connection withthis application and which are open to public inspection with thisspecification, and the contents of all such papers and documents areincorporated herein by reference.

1. A method for determining whether a subject with melanoma has anincreased risk of metastasis, the method comprising: (i) determining theexpression of Loricrin in a tissue sample obtained from the subjectusing a monoclonal antibody against Loricrin, wherein the tissue samplecomprises tissue overlying a primary melanoma; and (ii) comparing theexpression obtained in (i) with a reference tissue or levels obtainedtherefrom, wherein a decrease in the expression of Loricrin in thetissue sample compared to the reference tissues or levels, or a loss ofexpression of Loricrin in the tissue sample, is indicative of anincreased risk of metastasis.
 2. (canceled)
 3. The method of claim 1,wherein: (a) the tissue sample comprises at least a portion of aperi-tumoral epidermis overlying the primary melanoma and the methodcomprises determining the expression of Loricrin in the stratum corneum;(b) the tissue sample comprises keratinocytes overlying the primarymelanoma; (c) the tissue sample is a biopsy, or a section thereof,obtained from the subject; and/or (d) determining the expression ofLoricrin in the tissue sample comprises: (i) contacting the tissuesample with the antibody against Loricrin; and (ii) visualising theantibody in the tissue sample with a reagent that generates a detectablesignal.
 4. The method of claim 1, wherein the antibody specificallybinds to: (i) amino acids 2-20 of human Loricrin (SEQ ID NO:42); (ii)amino acids 73-91 of human Loricrin (SEQ ID NO:42); (iii) amino acids95-109, 120-134, 135-148 and/or 168-182 of human Loricrin (SEQ IDNO:42); (iv) amino acids 262-280 of human Loricrin (SEQ ID NO: 42); (v)amino acids 279-297 of human Loricrin (SEQ ID NO: 42); and/or (vi) aminoacids 298-308 of human Loricrin (SEQ ID NO: 42).
 5. The method of claim1, wherein the antibody against Loricrin comprises the following heavychain variable domain complementarity determining regions (CDRs): (a)HCDR1 comprising the amino acid sequence of SEQ ID NO: 22; (b) HCDR2comprising the amino acid sequence of SEQ ID NO: 23; and (c) HCDR3comprising the amino acid sequence of SEQ ID NO: 24; and optionallyfurther comprises the following heavy chain variable domain frameworkregions (FRs): (i) HCFR1 comprising the amino acid sequence of SEQ IDNO: 28; (ii) HCFR2 comprising the amino acid sequence of SEQ ID NO: 29;(iii) HCFR3 comprising the amino acid sequence of SEQ ID NO 30; and (iv)HCFR4 comprising the amino acid sequence of SEQ ID NO:
 31. 6. (canceled)7. The method of claim 1, wherein the antibody against Loricrincomprises the following light chain variable domain complementaritydetermining regions (CDRs): (a) LCDR1 comprising the amino acid sequenceof SEQ ID NO: 25; (b) LCDR2 comprising the amino acid sequence of SEQ IDNO: 26; and (c) LCDR3 comprising the amino acid sequence of SEQ ID NO:27; and optionally further comprises the following light chain variabledomain FRs: (i) LCFR1 comprising the amino acid sequence of SEQ ID NO:32; (ii) LCFR2 comprising the amino acid sequence of SEQ ID NO: 33;(iii) LCFR3 comprising the amino acid sequence of SEQ ID NO: 34; and(iv) LCFR4 comprising the amino acid sequence of SEQ ID NO:
 35. 8.(canceled)
 9. The method of claim 1, wherein the antibody againstLoricrin comprises: (a) a VH sequence of SEQ ID NO: 36, or a sequencehaving at least 95% sequence identity to the amino acid sequence of SEQID NO: 36; and/or: (b) a VL sequence of SEQ ID NO: 37, or a sequencehaving at least 95% sequence identity to the amino acid sequence of SEQID NO: 37; or (c) a Fab fragment comprising a Fd chain sequence of SEQID NO:38 and/or a light chain sequence of SEQ ID NO:39; and/or (d) thefollowing CDRs: (i) HCDR1 comprising the amino acid sequence of SEQ IDNO: 22; (ii) HCDR2 comprising the amino acid sequence of SEQ ID NO: 23;(iii) HCDR3 comprising the amino acid sequence of SEQ ID NO: 24; (iv)LCDR1 comprising the amino acid sequence of SEQ ID NO: 25; (v) LCDR2comprising the amino acid sequence of SEQ ID NO: 26; and (vi) LCDR3comprising the amino acid sequence of SEQ ID NO: 27; optionally whereinthe antibody against Loricrin further comprises the following heavychain variable domain and light chain variable domain FRs: (vii) HCFR1comprising the amino acid sequence of SEQ ID NO: 28; (viii) HCFR2comprising the amino acid sequence of SEQ ID NO: 29; (ix) HCFR3comprising the amino acid sequence of SEQ ID NO: 30; (x) HCFR4comprising the amino acid sequence of SEQ ID NO: 31; (xi) LCFR1comprising the amino acid sequence of SEQ ID NO: 32; (xii) LCFR2comprising the amino acid sequence of SEQ ID NO: 33; (xiii) LCFR3comprising the amino acid sequence of SEQ ID NO: 34; and (xiv) LCFR4comprising the amino acid sequence of SEQ ID NO:
 35. 10.-14. (canceled)15. Use of the antibody against Loricrin as claim 4 for determiningwhether a subject with melanoma has an increased risk of metastasis. 16.An antibody that competes for binding to Loricrin or binds to the sameepitope as the antibody as defined in claim
 4. 17. (canceled)
 18. Amethod for determining a treatment regime for a subject suffering frommelanoma, the method comprising: (i) determining the expression ofLoricrin in a tissue sample obtained from the subject using a monoclonalantibody against Loricrin, wherein the tissue sample comprises tissueoverlying a primary melanoma; and (ii) comparing the expression obtainedin (i) with a reference tissue or levels obtained therefrom, and (iii)(a) if expression of Loricrin is normal, following a normal recognizedcare pathway, or (b) if expression of Loricrin is decreased or lost,treating the subject with a systemic anti-cancer treatment regime. 19.The method of claim 18, wherein determining the expression of Loricrinin the tissue sample comprises: (a) contacting the tissue sample withthe antibody against Loricrin; and (b) visualising the antibody in thetissue sample with a reagent that generates a detectable signal.
 20. Themethod of claim 18, wherein: (i) the tissue sample comprises at least aportion of a peri-tumoral epidermis overlying the primary melanoma andthe method comprises determining the expression of Loricrin in thestratum corneum; (ii) the antibody against Loricrin binds to: (a) aminoacids 2-20 of human Loricrin (SEQ ID NO:42); (b) amino acids 73-91 ofhuman Loricrin (SEQ ID NO:42); (c) amino acids 95-109, 120-134, 135-148and/or 168-182 of human Loricrin (SEQ ID NO:42); (d) amino acids 262-280of human Loricrin (SEQ ID NO: 42); (e) amino acids 279-297 of humanLoricrin (SEQ ID NO: 42); (f) amino acids 298-308 of human Loricrin (SEQID NO: 42); (iii) the systemic anti-cancer treatment regime is forpreventing, inhibiting or delaying metastasis or decreasing the risk ofmetastasis in the subject; and/or (iv) the systemic anti-cancertreatment regime comprises administering a therapeutic agent to thesubject. 21.-23. (canceled)
 24. A method of treating a subject sufferingfrom melanoma, the method comprising: (i) determining the expression ofLoricrin in a tissue sample obtained from the subject using a monoclonalantibody against Loricrin, wherein the tissue sample comprises tissueoverlying a primary melanoma; and (ii) comparing the expression obtainedin (i) with a reference tissue or levels obtained therefrom, and ifthere is a decrease in the expression of Loricrin in the tissue samplecompared to the reference tissue or levels, or a loss of expression ofLoricrin, administering a therapeutic agent to the subject.
 25. Themethod of claim 24, wherein determining the expression of Loricrin inthe tissue sample comprises: (a) contacting the tissue sample with theantibody against Loricrin; and (b) visualising the antibody in thetissue sample with a reagent that generates a detectable signal.
 26. Themethod of claim 24, wherein: (i) the tissue sample comprises at least aportion of a peri-tumoral epidermis overlying the primary melanoma andthe method comprises determining the expression of Loricrin in thestratum corneum; (ii) the antibody against Loricrin specifically bindsto: (a) amino acids 2-20 of human Loricrin (SEQ ID NO:42); (b) aminoacids 73-91 of human Loricrin (SEQ ID NO:42); (c) amino acids 95-109,120-134, 135-148 and/or 168-182 of human Loricrin (SEQ ID NO:42); (d)amino acids 262-280 of human Loricrin (SEQ ID NO: 42); (e) amino acids279-297 of human Loricrin (SEQ ID NO: 42); and/or (iii) the therapeuticagent is: (a) a chemotherapeutic agent, optionally selected fromDacarbazine (DTIC), Temozolomide, Nab-paclitaxel, Paclitaxel, Carmustine(BCNU), Cisplatin, Carboplatin, Vinblastine, interleukin 2, interferonalpha, antibodies and B-Raf inhibitors, or (b) the chemotherapeuticagent is a biological agent, optionally selected from: (1) nivolumaband/or ipilimumab; (2) dabrafenib and/or trametinib; (3) vemurafeniband/or cobimetinib; and/or (4) any combination thereof. 27.-29.(canceled)
 30. The method of claim 24, wherein the therapeutic agent isadministered to the subject no more than 12 weeks after determining adecrease or loss of expression of Loricrin in the tissue sample.
 31. Themethod of claim 24, wherein: (a) the reference levels are levels ofLoricrin expression that are characteristic of normal tissue; (b) thereference tissue comprises normal tissue, optionally wherein the normaltissue is epidermis from a site which does not include a primarymelanoma; (c) the reference tissue is an internal reference; (d) thenormal tissue is from a site adjacent to the primary melanoma; (e) theexpression of Loricrin in the tissue sample is from about 25% to about75% of the respective reference level; (f) the expression of Loricrin inthe tissue sample is less than about 25% of the respective referencelevel; and/or (g) the expression of Loricrin in the tissue sample isdetermined by visual assessment or by an automatic slide scanner. 32.(canceled)
 33. A method of treating a subject suffering from melanoma,the method comprising administering a therapeutic agent to the subject,wherein the subject has been identified as having decreased or a loss ofexpression of Loricrin according to claim 1, wherein optionally themethod is for preventing, inhibiting or delaying metastasis ordecreasing the risk of metastasis in the subject.
 34. (canceled)
 35. Themethod of claim 33, wherein the subject, prior to identification, wasineligible for therapeutic agent treatment and/or wherein thetherapeutic agent is administered to the subject no more than 12 weeksafter the subject has been identified as having decreased or loss ofexpression of Loricrin in the tissue sample, wherein optionally thetherapeutic agent is a chemotherapeutic agent, optionally selected from:(i) Dacarbazine (DTIC), Temozolomide, Nab-paclitaxel, Paclitaxel,Carmustine (BCNU), Cisplatin, Carboplatin, Vinblastine, interleukin 2,interferon alpha, antibodies and B-Raf inhibitors; or (ii) nivolumaband/or ipilimumab; dabrafenib and/or trametinib; vemurafenib and/orcobimetinib and/or any combination thereof. 36.-38. (canceled)
 39. Themethod of claim 1, wherein: (a) the subject is suffering from AmericanJoint Commission on Cancer (AJCC) stage 1 or stage 2 or stage 3 or stage4 melanoma; (b) the subject is suffering from AJCC stage 1a, stage 1b,stage 2a, stage 2b or stage 2c melanoma; and/or (c) the subject has anulcerated melanoma.
 40. An in vitro assay for predicting an increasedrisk of metastasis in a subject suffering from melanoma, the assaycomprising: contacting a tissue sample obtained from the subject with anantibody against Loricrin as defined in claim 4, wherein the tissuesample comprises tissue overlying a primary melanoma and the presence ofLoricrin creates an Loricrin-antibody complex; and detecting and/orquantifying the Loricrin-antibody complex, wherein optionally: (a) thetissue sample comprises at least a portion of a peri-tumoral epidermisoverlying the primary melanoma and the Loricrin-antibody complex isdetected in the in the stratum corneum of the epidermis; and/or (b) theLoricrin-antibody complex is detected and/or quantified by visualassessment or by an automated slide scanner.
 41. (canceled)
 42. A kitfor predicting an increased risk of developing metastasis of a subjectsuffering from melanoma, the kit comprising an antibody against Loricrinas defined in claim 4, wherein optionally the kit further comprises atleast one capture agent, wherein the at least one capture agentcomprises a detection moiety and/or a binding moiety specific for theantibody against Loricrin.
 43. (canceled)